Cleavage of nucleic acids

ABSTRACT

The present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. Enzymes, including 5′ nucleases and 3′ exonucleases, are used to detect and identify nucleic acids derived from microorganisms. Methods are provided which allow for the detection and identification of bacterial and viral pathogens in a sample.

The present application is a Continuation of U.S. application Ser. No.09/941,193, filed Aug. 28, 2001, now U.S. Pat. No. 7,067,643, which is aDivisional application of U.S. App. Ser. No. 09/655,378, filed Sep. 5,2000, now U.S. Pat. No. 6,673,616, which is a Continuation of U.S.application Ser. No. 08/520,946, filed Aug. 30, 1995, now U.S. Pat. No.6,372,424, which is a Continuation-in-Part application of U.S.application Ser. No. 08/484,956, filed Jun. 7, 1995, now U.S. Pat. No.5,843,654, which is a Continuation-in-Part of U.S. App. Ser. No.08/402,601, filed Mar. 9, 1995, now abandoned, which is aContinuation-in-Part of U.S. application Ser. No. 08/337,164, filed Nov.9, 1994, now abandoned, which is a Continuation-in-Part of U.S.application Ser. No. 08/254,359, filed Jun. 6, 1994, now U.S. Pat. No.5,614,402.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for treatingnucleic acid, and in particular, methods and compositions for detectionand characterization of nucleic acid sequences and sequence changes.

BACKGROUND OF THE INVENTION

The detection and characterization of specific nucleic acid sequencesand sequence changes have been utilized to detect the presence of viralor bacterial nucleic acid sequences indicative of an infection, thepresence of variants or alleles of mammalian genes associated withdisease and cancers, and the identification of the source of nucleicacids found in forensic samples, as well as in paternity determinations.

Various methods are known in the art which may be used to detect andcharacterize specific nucleic acid sequences and sequence changes.Nonetheless, as nucleic acid sequence data of the human genome, as wellas the genomes of pathogenic organisms accumulates, the demand for fast,reliable, cost-effective and user-friendly tests for specific sequencescontinues to grow. Importantly, these tests must be able to create adetectable signal from a very low copy number of the sequence ofinterest. The following discussion examines three levels of nucleic aciddetection currently in use: I. Signal Amplification Technology fordetection of rare sequences; II. Direct Detection Technology fordetection of higher copy number sequences; and III. Detection of UnknownSequence Changes for rapid screening of sequence changes anywhere withina defined DNA fragment.

I. Signal Amplification Technology Methods for Amplification

The “Polymerase Chain Reaction” (PCR) comprises the first generation ofmethods for nucleic acid amplification. However, several other methodshave been developed that employ the same basis of specificity, butcreate signal by different amplification mechanisms. These methodsinclude the “Ligase Chain Reaction” (LCR), “Self-Sustained SyntheticReaction” (3SR/NASBA), and “Qβ-Replicase” (Qβ).

Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR), as described in U.S. Pat. Nos.4,683,195 and 4,683,202 to Mullis and Mullis et al., describe a methodfor increasing the concentration of a segment of target sequence in amixture of genomic DNA without cloning or purification. This technologyprovides one approach to the problems of low target sequenceconcentration. PCR can be used to directly increase the concentration ofthe target to an easily detectable level. This process for amplifyingthe target sequence involves introducing a molar excess of twooligonucleotide primers which are complementary to their respectivestrands of the double-stranded target sequence to the DNA mixturecontaining the desired target sequence. The mixture is denatured andthen allowed to hybridize. Following hybridization, the primers areextended with polymerase so as to form complementary strands. The stepsof denaturation, hybridization, and polymerase extension can be repeatedas often as needed, in order to obtain relatively high concentrations ofa segment of the desired target sequence.

The length of the segment of the desired target sequence is determinedby the relative positions of the primers with respect to each other,and, therefore, this length is a controllable parameter. Because thedesired segments of the target sequence become the dominant sequences(in terms of concentration) in the mixture, they are said to be“PCR-amplified.”

Ligase Chain Reaction (LCR or LAR)

The ligase chain reaction (LCR; sometimes referred to as “LigaseAmplification Reaction” (LAR) described by Barany, Proc. Natl. Acad.Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wuand Wallace, Genomics 4:560 (1989) has developed into a well-recognizedalternative method for amplifying nucleic acids. In LCR, fouroligonucleotides, two adjacent oligonucleotides which uniquely hybridizeto one strand of target DNA, and a complementary set of adjacentoligonucleotides, which hybridize to the opposite strand are mixed andDNA ligase is added to the mixture. Provided that there is completecomplementarity at the junction, ligase will covalently link each set ofhybridized molecules. Importantly, in LCR, two probes are ligatedtogether only when they base-pair with sequences in the target sample,without gaps or mismatches. Repeated cycles of denaturation,hybridization and ligation amplify a short segment of DNA. LCR has alsobeen used in combination with PCR to achieve enhanced detection ofsingle-base changes. Segev, PCT Public. No. WO9001069 A1 (1990).However, because the four oligonucleotides used in this assay can pairto form two short ligatable fragments, there is the potential for thegeneration of target-independent background signal. The use of LCR formutant screening is limited to the examination of specific nucleic acidpositions.

Self-Sustained Synthetic Reaction (3SR/NASBA)

The self-sustained sequence replication reaction (3SR) (Guatelli et al.,Proc. Natl. Acad. Sci., 87:1874-1878 (1990), with an erratum at Proc.Natl. Acad. Sci., 87:7797 (1990)) is a transcription-based in vitroamplification system (Kwok et al., Proc. Natl. Acad. Sci., 86:1173-1177(1989)) that can exponentially amplify RNA sequences at a uniformtemperature. The amplified RNA can then be utilized for mutationdetection (Fahy et al., PCR Meth. Appl., 1:25-33 (1991)). In thismethod, an oligonucleotide primer is used to add a phage RNA polymerasepromoter to the 5′ end of the sequence of interest. In a cocktail ofenzymes and substrates that includes a second primer, reversetranscriptase, RNase H, RNA polymerase and ribo- and deoxyribonucleosidetriphosphates, the target sequence undergoes repeated rounds oftranscription, cDNA synthesis and second-strand synthesis to amplify thearea of interest. The use of 3SR to detect mutations is kineticallylimited to screening small segments of DNA (e.g., 200-300 base pairs).

Q-Beta (Qβ) Replicase

In this method, a probe which recognizes the sequence of interest isattached to the replicatable RNA template for Qβ replicase. A previouslyidentified major problem with false positives resulting from thereplication of unhybridized probes has been addressed through use of asequence-specific ligation step. However, available thermostable DNAligases are not effective on this RNA substrate, so the ligation must beperformed by T4 DNA ligase at low temperatures (37° C.). This preventsthe use of high temperature as a means of achieving specificity as inthe LCR, the ligation event can be used to detect a mutation at thejunction site, but not elsewhere.

Table 1 below, lists some of the features desirable for systems usefulin sensitive nucleic acid diagnostics, and summarizes the abilities ofeach of the major amplification methods (See also, Landgren, Trends inGenetics 9:199 (1993)).

A successful diagnostic method must be very specific. A straight-forwardmethod of controlling the specificity of nucleic acid hybridization isby controlling the temperature of the reaction. While the 3SR/NASBA, andQβ systems are all able to generate a large quantity of signal, one ormore of the enzymes involved in each cannot be used at high temperature(i.e., >55° C.). Therefore the reaction temperatures cannot be raised toprevent non-specific hybridization of the probes. If probes areshortened in order to make them melt more easily at low temperatures,the likelihood of having more than one perfect match in a complex genomeincreases. For these reasons, PCR and LCR currently dominate theresearch field in detection technologies.

TABLE 1 METHOD: PCR & 3SR FEATURE PCR LCR LCR NASBA Qβ AmplifiesTarget + + + + Recognition of Independent + + + + + Sequences RequiredPerformed at High Temp. + + Operates at Fixed Temp. + + ExponentialAmplification + + + + + Generic Signal Generation + Easily Automatable

The basis of the amplification procedure in the PCR and LCR is the factthat the products of one cycle become usable templates in all subsequentcycles, consequently doubling the population with each cycle. The finalyield of any such doubling system can be expressed as: (1+X)^(n)=y,where “X” is the mean efficiency (percent copied in each cycle), “n” isthe number of cycles, and “y” is the overall efficiency, or yield of thereaction (Mullis, PCR Methods Applic., 1:1 (1991)). If every copy of atarget DNA is utilized as a template in every cycle of a polymerasechain reaction, then the mean efficiency is 100%. If 20 cycles of PCRare performed, then the yield will be 2²⁰, or 1,048,576 copies of thestarting material. If the reaction conditions reduce the mean efficiencyto 85%, then the yield in those 20 cycles will be only 1.85²⁰, or220,513 copies of the starting material. In other words, a PCR runningat 85% efficiency will yield only 21% as much final product, compared toa reaction running at 100% efficiency. A reaction that is reduced to 50%mean efficiency will yield less than 1% of the possible product.

In practice, routine polymerase chain reactions rarely achieve thetheoretical maximum yield, and PCRs are usually run for more than 20cycles to compensate for the lower yield. At 50% mean efficiency, itwould take 34 cycles to achieve the million-fold amplificationtheoretically possible in 20, and at lower efficiencies, the number ofcycles required becomes prohibitive. In addition, any backgroundproducts that amplify with a better mean efficiency than the intendedtarget will become the dominant products.

Also, many variables can influence the mean efficiency of PCR, includingtarget DNA length and secondary structure, primer length and design,primer and dNTP concentrations, and buffer composition, to name but afew. Contamination of the reaction with exogenous DNA (e.g., DNA spilledonto lab surfaces) or cross-contamination is also a major consideration.Reaction conditions must be carefully optimized for each differentprimer pair and target sequence, and the process can take days, even foran experienced investigator. The laboriousness of this process,including numerous technical considerations and other factors, presentsa significant drawback to using PCR in the clinical setting. Indeed, PCRhas yet to penetrate the clinical market in a significant way. The sameconcerns arise with LCR, as LCR must also be optimized to use differentoligonucleotide sequences for each target sequence. In addition, bothmethods require expensive equipment, capable of precise temperaturecycling.

Many applications of nucleic acid detection technologies, such as instudies of allelic variation, involve not only detection of a specificsequence in a complex background, but also the discrimination betweensequences with few, or single, nucleotide differences. One method forthe detection of allele-specific variants by PCR is based upon the factthat it is difficult for Taq polymerase to synthesize a DNA strand whenthere is a mismatch between the template strand and the 3′ end of theprimer. An allele-specific variant may be detected by the use of aprimer that is perfectly matched with only one of the possible alleles;the mismatch to the other allele acts to prevent the extension of theprimer, thereby preventing the amplification of that sequence. Thismethod has a substantial limitation in that the base composition of themismatch influences the ability to prevent extension across themismatch, and certain mismatches do not prevent extension or have only aminimal effect (Kwok et al., Nucl. Acids Res., 18:999 (1990)).

A similar 3′-mismatch strategy is used with greater effect to preventligation in the LCR (Barany, PCR Meth. Applic., 1:5 (1991)). Anymismatch effectively blocks the action of the thermostable ligase, butLCR still has the drawback of target-independent background ligationproducts initiating the amplification. Moreover, the combination of PCRwith subsequent LCR to identify the nucleotides at individual positionsis also a clearly cumbersome proposition for the clinical laboratory.

II. Direct Detection Technology

When a sufficient amount of a nucleic acid to be detected is available,there are advantages to detecting that sequence directly, instead ofmaking more copies of that target, (e.g., as in PCR and LCR). Mostnotably, a method that does not amplify the signal exponentially is moreamenable to quantitative analysis. Even if the signal is enhanced byattaching multiple dyes to a single oligonucleotide, the correlationbetween the final signal intensity and amount of target is direct. Sucha system has an additional advantage that the products of the reactionwill not themselves promote further reaction, so contamination of labsurfaces by the products is not as much of a concern. Traditionalmethods of direct detection including Northern and Southern blotting andRNase protection assays usually require the use of radioactivity and arenot amenable to automation. Recently devised techniques have sought toeliminate the use of radioactivity and/or improve the sensitivity inautomatable formats. Two examples are the “Cycling Probe Reaction”(CPR), and “Branched DNA” (bDNA)

The cycling probe reaction (CPR) (Duck et al., BioTech., 9:142 (1990)),uses a long chimeric oligonucleotide in which a central portion is madeof RNA while the two termini are made of DNA. Hybridization of the probeto a target DNA and exposure to a thermostable RNase H causes the RNAportion to be digested. This destabilizes the remaining DNA portions ofthe duplex, releasing the remainder of the probe from the target DNA andallowing another probe molecule to repeat the process. The signal, inthe form of cleaved probe molecules, accumulates at a linear rate. Whilethe repeating process increases the signal, the RNA portion of theoligonucleotide is vulnerable to RNases that may carried through samplepreparation.

Branched DNA (bDNA), described by Urdea et al., Gene 61:253-264 (1987),involves oligonucleotides with branched structures that allow eachindividual oligonucleotide to carry 35 to 40 labels (e.g., alkalinephosphatase enzymes). While this enhances the signal from ahybridization event, signal from non-specific binding is similarlyincreased.

III. Detection of Unknown Sequence Changes

The demand for tests which allow the detection of specific nucleic acidsequences and sequence changes is growing rapidly in clinicaldiagnostics. As nucleic acid sequence data for genes from humans andpathogenic organisms accumulates, the demand for fast, cost-effective,and easy-to-use tests for as yet unknown mutations within specificsequences is rapidly increasing.

A handful of methods have been devised to scan nucleic acid segments formutations. One option is to determine the entire gene sequence of eachtest sample (e.g., a bacterial isolate). For sequences underapproximately 600 nucleotides, this may be accomplished using amplifiedmaterial (e.g., PCR reaction products). This avoids the time and expenseassociated with cloning the segment of interest. However, specializedequipment and highly trained personnel are required, and the method istoo labor-intense and expensive to be practical and effective in theclinical setting.

In view of the difficulties associated with sequencing, a given segmentof nucleic acid may be characterized on several other levels. At thelowest resolution, the size of the molecule can be determined byelectrophoresis by comparison to a known standard run on the same gel. Amore detailed picture of the molecule may be achieved by cleavage withcombinations of restriction enzymes prior to electrophoresis, to allowconstruction of an ordered map. The presence of specific sequenceswithin the fragment can be detected by hybridization of a labeled probe,or the precise nucleotide sequence can be determined by partial chemicaldegradation or by primer extension in the presence of chain-terminatingnucleotide analogs.

For detection of single-base differences between like sequences, therequirements of the analysis are often at the highest level ofresolution. For cases in which the position of the nucleotide inquestion is known in advance, several methods have been developed forexamining single base changes without direct sequencing. For example, ifa mutation of interest happens to fall within a restriction recognitionsequence, a change in the pattern of digestion can be used as adiagnostic tool (e.g., restriction fragment length polymorphism (RFLP)analysis).

Single point mutations have been also detected by the creation ordestruction of RFLPs. Mutations are detected and localized by thepresence and size of the RNA fragments generated by cleavage at themismatches. Single nucleotide mismatches in DNA heteroduplexes are alsorecognized and cleaved by some chemicals, providing an alternativestrategy to detect single base substitutions, generically named the“Mismatch Chemical Cleavage” (MCC) (Gogos et al., Nucl. Acids Res.,18:6807-6817 (1990)). However, this method requires the use of osmiumtetroxide and piperidine, two highly noxious chemicals which are notsuited for use in a clinical laboratory.

RFLP analysis suffers from low sensitivity and requires a large amountof sample. When RFLP analysis is used for the detection of pointmutations, it is, by its nature, limited to the detection of only thosesingle base changes which fall within a restriction sequence of a knownrestriction endonuclease. Moreover, the majority of the availableenzymes have 4 to 6 base-pair recognition sequences, and cleave toofrequently for many large-scale DNA manipulations (Eckstein and Lilley(eds.), Nucleic Acids and Molecular Biology, vol. 2, Springer-Verlag,Heidelberg (1988)). Thus, it is applicable only in a small fraction ofcases, as most mutations do not fall within such sites.

A handful of rare-cutting restriction enzymes with 8 base-pairspecificities have been isolated and these are widely used in geneticmapping, but these enzymes are few in number, are limited to therecognition of G+C-rich sequences, and cleave at sites that tend to behighly clustered (Barlow and Lehrach, Trends Genet., 3:167 (1987)).Recently, endonucleases encoded by group I introns have been discoveredthat might have greater than 12 base-pair specificity (Perlman andButow, Science 246:1106 (1989)), but again, these are few in number.

If the change is not in a recognition sequence, then allele-specificoligonucleotides (ASOs), can be designed to hybridize in proximity tothe unknown nucleotide, such that a primer extension or ligation eventcan be used as the indicator of a match or a mis-match. Hybridizationwith radioactively labeled allelic specific oligonucleotides (ASO) alsohas been applied to the detection of specific point mutations (Conner etal., Proc. Natl. Acad. Sci., 80:278-282 (1983)). The method is based onthe differences in the melting temperature of short DNA fragmentsdiffering by a single nucleotide. Stringent hybridization and washingconditions can differentiate between mutant and wild-type alleles. TheASO approach applied to PCR products also has been extensively utilizedby various researchers to detect and characterize point mutations in rasgenes (Vogelstein et al., N. Eng. J. Med., 319:525-532 (1988); and Farret al., Proc. Natl. Acad. Sci., 85:1629-1633 (1988)), and gsp/giponcogenes (Lyons et al., Science 249:655-659 (1990)). Because of thepresence of various nucleotide changes in multiple positions, the ASOmethod requires the use of many oligonucleotides to cover all possibleoncogenic mutations.

With either of the techniques described above (i.e., RFLP and ASO), theprecise location of the suspected mutation must be known in advance ofthe test. That is to say, they are inapplicable when one needs to detectthe presence of a mutation of an unknown character and position within agene or sequence of interest.

Two other methods rely on detecting changes in electrophoretic mobilityin response to minor sequence changes. One of these methods, termed“Denaturing Gradient Gel Electrophoresis” (DGGE) is based on theobservation that slightly different sequences will display differentpatterns of local melting when electrophoretically resolved on agradient gel. In this manner, variants can be distinguished, asdifferences in melting properties of homoduplexes versus heteroduplexesdiffering in a single nucleotide can detect the presence of mutations inthe target sequences because of the corresponding changes in theirelectrophoretic mobilities. The fragments to be analyzed, usually PCRproducts, are “clamped” at one end by a long stretch of G-C base pairs(30-80) to allow complete denaturation of the sequence of interestwithout complete dissociation of the strands. The attachment of a GC“clamp” to the DNA fragments increases the fraction of mutations thatcan be recognized by DGGE (Abrams et al., Genomics 7:463-475 (1990)).Attaching a GC clamp to one primer is critical to ensure that theamplified sequence has a low dissociation temperature (Sheffield et al.,Proc. Natl. Acad. Sci., 86:232-236 (1989); and Lerman and Silverstein,Meth. Enzymol., 155:482-501 (1987)). Modifications of the technique havebeen developed, using temperature gradients (Wartell et al., Nucl. AcidsRes., 18:2699-2701 (1990)), and the method can be also applied toRNA:RNA duplexes (Smith et al., Genomics 3:217-223 (1988)).

Limitations on the utility of DGGE include the requirement that thedenaturing conditions must be optimized for each type of DNA to betested. Furthermore, the method requires specialized equipment toprepare the gels and maintain the needed high temperatures duringelectrophoresis. The expense associated with the synthesis of theclamping tail on one oligonucleotide for each sequence to be tested isalso a major consideration. In addition, long running times are requiredfor DGGE. The long running time of DGGE was shortened in a modificationof DGGE called constant denaturant gel electrophoresis (CDGE) (Borrensenet al., Proc. Natl. Acad. Sci. USA 88:8405 (1991)). CDGE requires thatgels be performed under different denaturant conditions in order toreach high efficiency for the detection of unknown mutations.

An technique analogous to DGGE, termed temperature gradient gelelectrophoresis (TGGE), uses a thermal gradient rather than a chemcialdenaturant gradient (Scholz, et al., Hum. Mol. Genet. 2:2155 (1993)).TGGE requires the use of specialized equipment which can generate atemperature gradient perpendicularly oriented relative to the electricalfield. TGGE can detect mutations in relatively small fragments of DNAtherefore scanning of large gene segments requires the use of multiplePCR products prior to running the gel.

Another common method, called “Single-Strand Conformation Polymorphism”(SSCP) was developed by Hayashi, Sekya and colleagues (reviewed byHayashi, PCR Meth. Appl., 1:34-38, (1991)) and is based on theobservation that single strands of nucleic acid can take oncharacteristic conformations in non-denaturing conditions, and theseconformations influence electrophoretic mobility. The complementarystrands assume sufficiently different structures that one strand may beresolved from the other. Changes in sequences within the fragment willalso change the conformation, consequently altering the mobility andallowing this to be used as an assay for sequence variations (Orita, etal., Genomics 5:874-879, (1989)).

The SSCP process involves denaturing a DNA segment (e.g., a PCR product)that is labelled on both strands, followed by slow electrophoreticseparation on a non-denaturing polyacrylamide gel, so thatintra-molecular interactions can form and not be disturbed during therun. This technique is extremely sensitive to variations in gelcomposition and temperature. A serious limitation of this method is therelative difficulty encountered in comparing data generated in differentlaboratories, under apparently similar conditions.

The dideoxy fingerprinting (ddF) is another technique developed to scangenes for the presence of unknown mutations (Liu and Sommer, PCR MethodsAppli., 4:97 (1994)). The ddF technique combines components of Sangerdideoxy sequencing with SSCP. A dideoxy sequencing reaction is performedusing one dideoxy terminator and then the reaction products areelectrophoresised on nondenaturing polyacrylamide gels to detectalterations in mobility of the termination segments as in SSCP analysis.While ddF is an improvement over SSCP in terms of increased sensitivity,ddF requires the use of expensive dideoxynucleotides and this techniqueis still limited to the analysis of fragments of the size suitable forSSCP (i.e., fragments of 200-300 bases for optimal detection ofmutations).

In addition to the above limitations, all of these methods are limitedas to the size of the nucleic acid fragment that can be analyzed. Forthe direct sequencing approach, sequences of greater than 600 base pairsrequire cloning, with the consequent delays and expense of eitherdeletion sub-cloning or primer walking, in order to cover the entirefragment. SSCP and DGGE have even more severe size limitations. Becauseof reduced sensitivity to sequence changes, these methods are notconsidered suitable for larger fragments. Although SSCP is reportedlyable to detect 90% of single-base substitutions within a 200 base-pairfragment, the detection drops to less than 50% for 400 base pairfragments. Similarly, the sensitivity of DGGE decreases as the length ofthe fragment reaches 500 base-pairs. The ddF technique, as a combinationof direct sequencing and SSCP, is also limited by the relatively smallsize of the DNA that can be screened.

Clearly, there remains a need for a method that is less sensitive tosize so that entire genes, rather than gene fragments, may be analyzed.Such a tool must also be robust, so that data from different labs,generated by researchers of diverse backgrounds and skills will becomparable. Ideally, such a method would be compatible with“multiplexing,” (i.e., the simultaneous analysis of several molecules orgenes in a single reaction or gel lane, usually resolved from each otherby differential labelling or probing). Such an analytical procedurewould facilitate the use of internal standards for subsequent analysisand data comparison, and increase the productivity of personnel andequipment. The ideal method would also be easily automatable.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for treatingnucleic acid, and in particular, methods and compositions for detectionand characterization of nucleic acid sequences and sequence changes inmicrobial gene sequences. The present invention provides means forcleaving a nucleic acid cleavage structure in a site-specific manner. Inone embodiment, the means for cleaving is an enzyme capable of cleavingcleavage structures on a nucleic acid substrate, forming the basis of anovel method of detection of specific nucleic acid sequences. Thepresent invention contemplates use of the novel detection method for,among other uses, clinical diagnostic purposes, including but notlimited to detection and identification of pathogenic organisms.

In one embodiment, the present invention contemplates a DNA sequenceencoding a DNA polymerase altered in sequence (i.e., a “mutant” DNApolymerase) relative to the native sequence such that it exhibitsaltered DNA synthetic activity from that of the native (i.e., “wildtype”) DNA polymerase. With regard to the polymerase, a complete absenceof synthesis is not required; it is desired that cleavage reactionsoccur in the absence of polymerase activity at a level where itinterferes with the method. It is preferred that the encoded DNApolymerase is altered such that it exhibits reduced synthetic activityfrom that of the native DNA polymerase. In this manner, the enzymes ofthe invention are nucleases and are capable of cleaving nucleic acids ina structure-specific manner. Importantly, the nucleases of the presentinvention are capable of cleaving cleavage structures to create discretecleavage products.

The present invention contemplates nucleases from a variety of sources,including nucleases that are thermostable. Thermostable nucleases arecontemplated as particularly useful, as they are capable of operating attemperatures where nucleic acid hybridization is extremely specific,allowing for allele-specific detection (including single-basemismatches). In one embodiment, the thermostable 5′ nucleases areselected from the group consisting of altered polymerases derived fromthe native polymerases of various Thermus species, including, but notlimited to Thermus aquaticus, Thermus flavus and Thermus thermophilus.

The present invention utilizes such enzymes in methods for detection andcharacterization of nucleic acid sequences and sequence changes. Thepresent invention relates to means for cleaving a nucleic acid cleavagestructure in a site-specific manner. Nuclease activity is used to screenfor known and unknown mutations, including single base changes, innucleic acids.

In one embodiment, the present invention contemplates a process ormethod for identifying strains of microorganisms comprising the steps ofproviding a cleavage means and a nucleic acid substrate containingsequences derived from one or more microorganism; treating the nucleicacid substrate under conditions such that the substrate forms one ormore cleavage structures; and reacting the cleavage means with thecleavage structures so that one or more cleavage products are produced.In one embodiment of this invention, the cleavage means is an enzyme. Inone preferred embodiment, the enzyme is a nuclease. In an alternativepreferred embodiment, the nuclease is selected from the group consistingof CLEAVASE BN enzyme, Thermus aquaticus DNA polymerase, Thermusthermophilus DNA polymerase, Escherichia coli Exo III, and theSaccharomyces cerevisiae Rad1/Rad10 complex. It is also contemplatedthat the enzyme may have a portion of its amino acid sequence that ishomologous to a portion of the amino acid sequence of a thermostable DNApolymerase derived from a eubacterial thermophile, the latter beingselected from the group consisting of Thermus aquaticus, Thermus flavusand Thermus thermophilus.

It is contemplated that the nucleic acid substrate comprise a nucleotideanalog, including but not limited to the group comprising 7-deaza-dATP,7-deaza-dGTP and dUTP. In one embodiment, the nucleic acid substrate issubstantially single-stranded. It is not intended that the nucleic acidsubstrate be limited to any particular form, indeed, it is contemplatedthat the nucleic acid substrate is single stranded or double-strandedRNA or DNA.

In one embodiment of the present invention, the treating step comprisesrendering double-stranded nucleic acid substantially single-stranded,and exposing the single-stranded nucleic acid to conditions such thatthe single-stranded nucleic acid assumes a secondary or characteristicfolded structure. In one preferred embodiment, double-stranded nucleicacid is rendered substantially single-stranded by increased temperature.

In an alternative embodiment, the method of the present inventionfurther comprises the step of detecting one or more cleavage products.

It is contemplated that the microorganism(s) of the present invention beselected from a variety of microorganisms. It is not intended that thepresent invention be limited to any particular type of microorganism.Rather, it is intended that the present invention be used with organismsincluding, but not limited to, bacteria, fungi, protozoa, ciliates, andviruses. It is not intended that the microorganisms be limited to aparticular genus, species, strain, or serotype. Indeed, it iscontemplated that the bacteria be selected from the group including, butnot limited to members of the genera Campylobacter, Escherichia,Mycobacterium, Salmonella, Shigella, and Staphylococcus. In onepreferred embodiment, the microorganism(s) comprise strains ofmulti-drug resistant Mycobacterium tuberculosis. It is also contemplatedthat the present invention be used with viruses, including but notlimited to hepatitis C virus and simian immunodeficiency virus.

Another embodiment of the present invention contemplates a method fordetecting and identifying strains of microorganisms, comprising thesteps of extracting nucleic acid from a sample suspected of containingone or more microorganisms; and contacting the extracted nucleic acidwith a cleavage means under conditions such that the extracted nucleicacid forms one or more secondary structures, and the cleavage meanscleaves the secondary structures to produce one or more cleavageproducts.

In one embodiment, the method further comprises the step of separatingthe cleavage products. In yet another embodiment, the method furthercomprises the step of detecting the cleavage products.

In one preferred embodiment, the present invention further comprisescomparing the detected cleavage products generated from cleavage of theextracted nucleic acid isolated from the sample with separated cleavageproducts generated by cleavage of nucleic acids derived from one or morereference microorganisms. In such a case the sequence of the nucleicacids from one or more reference microorganisms may be related butdifferent (e.g., a wild type control for a mutant sequence or a known orpreviously characterized mutant sequence).

In an alternative preferred embodiment, the present invention furthercomprises the step of isolating a polymorphic locus from the extractednucleic acid after the extraction step, so as to generate a nucleic acidsubstrate, wherein the substrate is contacted with the cleavage means.In one embodiment, the isolation of a polymorphic locus is accomplishedby polymerase chain reaction amplification. In an alternate embodiment,the polymerase chain reaction is conducted in the presence of anucleotide analog, including but not limited to the group comprising7-deaza-dATP, 7-deaza-dGTP and dUTP. It is contemplated that thepolymerase chain reaction amplification will employ oligonucleotideprimers matching or complementary to consensus gene sequences derivedfrom the polymorphic locus. In one embodiment, the polymorphic locuscomprises a ribosomal RNA gene. In a particularly preferred embodiment,the ribosomal RNA gene is a 16S ribosomal RNA gene.

In one embodiment of this method, the cleavage means is an enzyme. Inone preferred embodiment, the enzyme is a nuclease. In a particularlypreferred embodiment, the nuclease is selected from the group including,but not limited to CLEAVASE BN enzyme, Thermus aquaticus DNA polymerase,Thermus thermophilus DNA polymerase, Escherichia coli Exo III, and theSaccharomyces cerevisiae Rad1/Rad10 complex. It is also contemplatedthat the enzyme may have a portion of its amino acid sequence that ishomologous to a portion of the amino acid sequence of a thermostable DNApolymerase derived from a eubacterial thermophile, the latter beingselected from the group consisting of Thermus aquaticus, Thermus flavusand Thermus thermophilus.

It is contemplated that the nucleic acid substrate of this method willcomprise a nucleotide analog, including but not limited to the groupcomprising 7-deaza-dATP, 7-deaza-dGTP and dUTP. In one embodiment, thenucleic acid substrate is substantially single-stranded. It is notintended that the nucleic acid substrate be limited to any particularform, indeed, it is contemplated that the nucleic acid substrate issingle stranded or double-stranded RNA or DNA.

In another embodiment of the present invention, the treating step of themethod comprises rendering double-stranded nucleic acid substantiallysingle-stranded, and exposing the single-stranded nucleic acid toconditions such that the single-stranded nucleic acid has secondarystructure. In one preferred embodiment, double-stranded nucleic acid isrendered substantially single-stranded by increased temperature.

It is contemplated that the microorganism(s) of the present invention beselected from a variety of microorganisms; it is not intended that thepresent invention be limited to any particular type of microorganism.Rather, it is intended that the present invention will be used withorganisms including, but not limited to, bacteria, fungi, protozoa,ciliates, and viruses. It is not intended that the microorganisms belimited to a particular genus, species, strain, or serotype. Indeed, itis contemplated that the bacteria be selected from the group comprising,but not limited to members of the genera Campylobacter, Escherichia,Mycobacterium, Salmonella, Shigella, and Staphylococcus. In onepreferred embodiment, the microorganism(s) comprise strains ofmulti-drug resistant Mycobacterium tuberculosis. It is also contemplatedthat the present invention be used with viruses, including but notlimited to hepatitis C virus and simian immunodeficiency virus.

In yet another embodiment, the present invention contemplates a methodfor treating nucleic acid comprising an oligonucleotide containingmicrobial gene sequences, comprising providing a cleavage means in asolution containing manganese and nucleic acid substrate containingmicrobial gene sequences; treating the nucleic acid substrate withincreased temperature such that the substrate is substantiallysingle-stranded; reducing the temperature under conditions such that thesingle-stranded substrate forms one or more cleavage structures;reacting the cleavage means with the cleavage structures so that one ormore cleavage products are produced; and detecting the one or morecleavage products produced by the method.

The present invention also contemplates a process for creating a recordreference library of genetic fingerprints characteristic (i.e.,diagnostic) of one or more alleles of the various microorganisms,comprising the steps of providing a cleavage means and nucleic acidsubstrate derived from microbial gene sequences; contacting the nucleicacid substrate with a cleavage means under conditions such that theextracted nucleic acid forms one or more secondary structures and thecleavage means cleaves the secondary structures, resulting in thegeneration of multiple cleavage products; separating the multiplecleavage products; and maintaining a testable record reference of theseparated cleavage products.

By the term “genetic fingerprint” it is meant that changes in thesequence of the nucleic acid (e.g., a deletion, insertion or a singlepoint substitution) alter the structures formed, thus changing thebanding pattern (i.e., the “fingerprint” or “bar code”) to reflect thedifference in the sequence, allowing rapid detection and identificationof variants.

The methods of the present invention allow for simultaneous analysis ofboth strands (e.g., the sense and antisense strands) and are ideal forhigh-level multiplexing. The products produced are amenable toqualitative, quantitative and positional analysis. The methods may beautomated and may be practiced in solution or in the solid phase (e.g.,on a solid support). The methods are powerful in that they allow foranalysis of longer fragments of nucleic acid than current methodologies.

DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a schematic of one embodiment of the detection methodof the present invention.

FIG. 1B provides a schematic of a second embodiment of the detectionmethod of the present invention.

FIGS. 2A-H is a comparison of the nucleotide structure of the DNAP genesisolated from Thermus aquaticus (SEQ ID NO:1), Thermus flavus (SEQ IDNO:2) and Thermus thermophilus (SEQ ID NO:3); the consensus sequence(SEQ ID NO:7) is shown at the top of each row.

FIGS. 3A-C is a comparison of the amino acid sequence of the DNAPisolated from Thermus aquaticus (SEQ ID NO:4), Thermus flavus (SEQ IDNO:5), and Thermus thermophilus (SEQ ID NO:6); the consensus sequence(SEQ ID NO:8) is shown at the top of each row.

FIGS. 4A-G are a set of diagrams of wild-type and synthesis-deficientDNAPTaq genes.

FIG. 5A depicts the wild-type Thermus flavus polymerase gene.

FIG. 5B depicts a synthesis-deficient Thermus flavus polymerase gene.

FIG. 6 depicts a structure (SEQ ID NOS:15 and 17) which cannot beamplified using DNAPTaq.

FIG. 7 is a ethidium bromide-stained gel demonstrating attempts toamplify a bifurcated duplex using either DNAPTaq or DNAPStf (Stoffel).

FIG. 8 is an autoradiogram of a gel analyzing the cleavage of abifurcated duplex by DNAPTaq and lack of cleavage by DNAPStf.

FIGS. 9A-B are a set of autoradiograms of gels analyzing cleavage orlack of cleavage upon addition of different reaction components andchange of incubation temperature during attempts to cleave a bifurcatedduplex with DNAPTaq.

FIGS. 10A-B are an autoradiogram displaying timed cleavage reactions,with and without primer.

FIGS. 11A-B are a set of autoradiograms of gels demonstrating attemptsto cleave a bifurcated duplex (with and without primer) with variousDNAPs.

FIG. 12A shows the substrates (SEQ ID NO:32) and oligonucleotides (SEQID NOS:18 and 19) used to test the specific cleavage of substrate DNAstargeted by pilot oligonucleotides.

FIG. 12B shows an autoradiogram of a gel showing the results of cleavagereactions using the substrates and oligonucleotides shown FIG. 12A.

FIG. 13A shows the substrate (SEQ ID NO:161) and oligonucleotide (SEQ IDNO:20) used to test the specific cleavage of a substrate RNA targeted bya pilot oligonucleotide.

FIG. 13B shows an autoradiogram of a gel showing the results of acleavage reaction using the substrate and oligonucleotide shown in FIG.13A.

FIGS. 14A-C is a diagram of vector pTTQ18 comprising SEQ ID NO:162.

FIGS. 15A-C is a diagram of vector pET-3c comprising SEQ ID NO:163.

FIGS. 16A-E depicts a set of molecules which are suitable substrates forcleavage by the 5′ nuclease activity of DNAPs.

FIG. 17 is an autoradiogram of a gel showing the results of a cleavagereaction run with synthesis-deficient DNAPs.

FIG. 18 is an autoradiogram of a PEI chromatogram resolving the productsof an assay for synthetic activity in synthesis-deficient DNAPTaqclones.

FIG. 19A depicts the substrate molecule (SEQ ID NO:164) used to test theability of synthesis-deficient DNAPs to cleave short hairpin structures.

FIG. 19B shows an autoradiogram of a gel resolving the products of acleavage reaction run using the substrate shown in FIG. 19A.

FIG. 20A shows the A- and T-hairpin molecules (SEQ ID NOS:23-24) used inthe trigger/detection assay.

FIG. 20B shows the sequence of the alpha primer (SEQ ID NO:25) used inthe trigger/detection assay.

FIG. 20C shows the structure of the cleaved A- (SEQ ID NO:28) andT-hairpin (SEQ ID NO:27) molecules.

FIG. 20D depicts the complementarity between the A- and T-hairpinmolecules.

FIG. 21 provides the complete 206-mer duplex sequence (SEQ ID NO:32)employed as a substrate for the 5′ nucleases of the present invention(shown within SEQ ID NO:165).

FIGS. 22A and B show the cleavage of linear nucleic acid substrates(based on the 206-mer of FIG. 21) by wild type DNAPs and 5′ nucleasesisolated from Thermus aquaticus and Thermus flavus.

FIG. 23 provides a detailed schematic corresponding to the of oneembodiment of the detection method of the present invention.

FIG. 24 shows the propagation of cleavage of the linear duplex nucleicacid structures of FIG. 23 by the 5′ nucleases of the present invention.

FIG. 25A shows the “nibbling” phenomenon detected with the DNAPs of thepresent invention.

FIG. 25B shows that the “nibbling” of FIG. 25A is 5′ nucleolyticcleavage and not phosphatase cleavage.

FIGS. 26A-B demonstrate that the “nibbling” phenomenon is duplexdependent.

FIG. 27 is a schematic showing how “nibbling” can be employed in adetection assay.

FIGS. 28A-B demonstrate that “nibbling” can be target directed.

FIG. 29 is a schematic showing the CFLP method of generating acharacteristic fingerprint from a nucleic acid substrate.

FIG. 30 shows an autoradiograph of a gel resolving the products ofcleavage reactions run in the presence of either MgCl₂ or MnCl₂.

FIG. 31 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on four similarly sized DNA substrates.

FIG. 32 shows an autoradiograph of a gel resolving the products ofcleavage reactions run using a wild-type and two mutant tyrosinase genesubstrates.

FIG. 33 shows an autoradiograph of a gel resolving the products ofcleavage reactions run using either a wild-type or mutant tyrosinasesubstrate varying in length from 157 nucleotides to 1.587 kb.

FIG. 34 shows an autoradiograph of a gel resolving the products ofcleavage reactions run in various concentrations of MnCl₂.

FIG. 35 shows an autoradiograph of a gel resolving the products ofcleavage reactions run in various concentrations of KCl.

FIG. 36 shows an autoradiograph of a gel resolving the products ofcleavage reactions run for different lengths of time.

FIG. 37 shows an autoradiograph of a gel resolving the products ofcleavage reactions run at different temperatures.

FIG. 38 shows an autoradiograph of a gel resolving the products ofcleavage reactions run using different amounts of the enzyme CLEAVASEBN.

FIG. 39 shows an autoradiograph of a gel resolving the products ofcleavage reactions run using four different preparations of the DNAsubstrate.

FIG. 40 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on either the sense or antisense strand of fourdifferent tyrosinase gene substrates.

FIG. 41 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on a wild-type β-globin substrate in twodifferent concentrations of KCl and at four different temperatures.

FIG. 42 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on two different mutant β-globin substrates infive different concentrations of KCl.

FIG. 43 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on a wild-type and three mutant β-globinsubstrates.

FIG. 44 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on an RNA substrate.

FIG. 45 shows an autoradiograph of a gel resolving the products ofcleavage reactions run using either the enzyme CLEAVASE BN or Taq DNApolymerase as the 5′ nuclease.

FIG. 46 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on a double-stranded DNA substrate to demonstratemultiplexing of the cleavage reaction.

FIG. 47 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on double-stranded DNA substrates consisting ofthe 419 and 422 mutant alleles derived from exon 4 of the humantyrosinase gene in the presence of various concentrations of MnCl₂.

FIG. 48 displays two traces representing two channel signals (JOE andFAM fluorescent dyes) for cleavage fragments derived from a cleavagereaction containing two differently labelled substrates (the wild-typeand 422 mutant substrates derived from exon 4 of the tyrosinase gene).The thin lines represent the JOE-labelled wild-type substrate and thethick lines represent the FAM-labelled 422 mutant substrate. Above thetracing is an autoradiograph of a gel resolving the products of cleavagereactions run on double-stranded DNA substrates consisting of thewild-type and 422 mutant alleles derived from exon 4 of the tyrosinasegene.

FIGS. 49A-G depicts the nucleotide sequence of six SIV LTR clonescorresponding to SEQ ID NOS:76-81.

FIG. 50 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on six different double-stranded SIV LTRsubstrates which contained a biotin label on the 5′ end of the (−)strand.

FIG. 51 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on six different double-stranded SIV LTRsubstrates which contained a biotin label on the 5′ end of the (+)strand.

FIG. 52 shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run in various concentrations ofNaCl.

FIG. 53 shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run in various concentrations of(NH₄)₂SO₄.

FIG. 54 shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run in increasing concentrations ofKCl.

FIG. 55 shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run in two concentrations of KCl forvarious periods of time.

FIG. 56 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on either the single-stranded or double-strandedform of the same substrate.

FIG. 57 shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions run in various concentrations of KCl.

FIG. 58 shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions run in various concentrations ofNaCl.

FIG. 59 shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions run in various concentrations of(NH₄)₂SO₄.

FIG. 60 shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions run for various lengths of time.

FIG. 61 shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions run using various amounts of CLEAVASEBN enzyme for either 5 seconds or 1 minute.

FIG. 62 shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions run at various temperatures.

FIG. 63 shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions run using various amounts of CLEAVASEBN enzyme.

FIG. 64A shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run in buffers having various pHs.

FIG. 64B shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run in buffers having a pH of either7.5 or 7.8.

FIG. 65A shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions run in buffers having a pH of either8.2 or 7.2.

FIG. 65B shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions run in buffers having a pH of either7.5 or 7.8.

FIG. 66 shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run in the presence of variousamounts of human genomic DNA.

FIG. 67 shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run using the Tfl DNA polymerase intwo different concentrations of KCl.

FIG. 68 shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run using the Tth DNA polymerase intwo different concentrations of KCl.

FIG. 69 shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run using the E. coli Exo III enzymein two different concentrations of KCl.

FIG. 70 shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run on three different tyrosinasegene substrates (SEQ ID NOS:47, 54 and 55) using either the Tth DNApolymerase, the E. coli Exo III enzyme or CLEAVASE BN enzyme.

FIG. 71 is a schematic drawing depicting the location of the 5′ and 3′cleavage sites on a cleavage structure.

FIG. 72 shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run on three different tyrosinasegene substrates (SEQ ID NOS:47, 54 and 55) using either CLEAVASE BNenzyme or the Rad1/Rad10 complex.

FIG. 73 shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions run on a wild-type and two mutantβ-globin substrates.

FIG. 74A shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run on a wild-type and three mutantβ-globin substrates.

FIG. 74B shows an autoradiograph of a gel resolving the products ofsingle-stranded cleavage reactions run on five mutant β-globinsubstrates.

FIG. 75 shows an autoradiograph of a gel resolving the products ofdouble-stranded cleavage reactions which varied the order of addition ofthe reaction components.

FIG. 76 depicts the organization of the human p53 gene; exons arerepresented by the solid black boxes and are labelled 1-11. Five hotspot regions are shown as a blow-up of the region spanning exons 5-8;the hot spot regions are labelled A, A′, B, C, and D.

FIG. 77 provides a schematic showing the use of a first 2-step PCRtechnique for the generation DNA fragments containing p53 mutations.

FIG. 78 provides a schematic showing the use of a second 2-step PCRtechnique for the generation DNA fragments containing p53 mutations.

FIG. 79 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on a wild-type and two mutant p53 substrates.

FIG. 80 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on a wild-type and three mutant p53 substrates.

FIG. 81 shows an autoradiograph of a gel resolving the products ofcleavage reactions run on a wild-type and a mutant p53 substrate wherethe mutant and wild-type substrates are present in variousconcentrations relative to one another.

FIG. 82 provides an alignment of HCV clones 1.1 (SEQ ID NO:121), HCV2.1(SEQ ID NO:122), HCV3.1 (SEQ ID NO:123), HCV4.2 (SEQ ID NO:124), HCV6.1(SEQ ID NO:125) and HCV7.1 (SEQ ID NO:126).

FIG. 83 shows a fluoroimager scan of a gel resolving the products ofcleavage reactions run on six double-stranded HCV substrates labeled oneither the sense or anti-sense strand.

FIG. 84 shows an autoradiogram of a gel resolving the products ofcleavage reactions run on a wild-type and two mutant M. tuberculosisrpoB substrates.

FIG. 85A shows a fluoroimager scan of a gel resolving the products ofcleavage reactions run on a wild-type and two mutant M. tuberculosisrpoB substrates prepared using either dTTP or dUTP.

FIG. 85B shows a fluoroimager scan of the gel shown in FIG. 85Afollowing a longer period of electrophoresis.

FIG. 86 shows an autoradiogram of a gel resolving the products ofcleavage reactions run on a wild-type and three mutant M. tuberculosiskatG substrates labeled on the sense strand.

FIG. 87 shows a fluoroimager scan of a gel resolving the products ofcleavage reactions run on a wild-type and three mutant M. tuberculosiskatG substrates labeled on the anti-sense strand.

FIGS. 88A-D shows the location of primers along the sequence of the E.coli rrsE gene (SEQ ID NO:158).

FIGS. 89A-F provides an alignment of the E. coli rrsE (SEQ ID NO:158),Cam. jejuni5 (SEQ ID NO:159), and Stp.aureus (SEQ ID NO:160) rRNA geneswith the location of consensus PCR rRNA primers indicated in bold type.

FIG. 90 shows a fluoroimager scan of a gel resolving the products ofcleavage reactions run on four bacterial 16S rRNA substrates.

FIG. 91A shows a fluoroimager scan of a gel resolving the products ofcleavage reactions run on five bacterial 16S rRNA substrates.

FIG. 91B shows bacterial a fluoroimager scan of a gel resolving theproducts of cleavage reactions run on five bacterial 16S rRNAsubstrates.

FIG. 92 shows bacterial a fluoroimager scan of a gel resolving theproducts of cleavage reactions run on various bacterial 16S rRNAsubstrates.

FIG. 93 shows bacterial a fluoroimager scan of a gel resolving theproducts of cleavage reactions run on eight bacterial 16S rRNAsubstrates.

FIG. 94 shows an autoradiogram of a gel resolving the products ofcleavage reactions run on a wild-type and mutant tyrosinase genesubstrates prepared using naturally occurring deoxynucleotides ordeoxynucleotide analogs.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence so long as the desiredenzymatic activity is retained.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

The term “recombinant DNA vector” as used herein refers to DNA sequencescontaining a desired coding sequence and appropriate DNA sequencesnecessary for the expression of the operably linked coding sequence in aparticular host organism. DNA sequences necessary for expression inprocaryotes include a promoter, optionally an operator sequence, aribosome binding site and possibly other sequences. Eukaryotic cells areknown to utilize promoters, polyadenlyation signals and enhancers.

The term “LTR” as used herein refers to the long terminal repeat foundat each end of a provirus (i.e., the integrated form of a retrovirus).The LTR contains numerous regulatory signals including transcriptionalcontrol elements, polyadenylation signals and sequences needed forreplication and integration of the viral genome. The viral LTR isdivided into three regions called U3, R and U5.

The U3 region contains the enhancer and promoter elements. The U5 regioncontains the polyadenylation signals. The R (repeat) region separatesthe U3 and U5 regions and transcribed sequences of the R region appearat both the 5′ and 3′ ends of the viral RNA.

The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably more than three, and usually more than ten. The exact sizewill depend on many factors, which in turn depends on the ultimatefunction or use of the oligonucleotide. The oligonucleotide may begenerated in any manner, including chemical synthesis, DNA replication,reverse transcription, or a combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide which is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

A primer is selected to be “substantially” complementary to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

“Hybridization” methods involve the annealing of a complementarysequence to the target nucleic acid (the sequence to be detected). Theability of two polymers of nucleic acid containing complementarysequences to find each other and anneal through base pairing interactionis a well-recognized phenomenon. The initial observations of the“hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960)have been followed by the refinement of this process into an essentialtool of modern biology. Nonetheless, a number of problems have preventedthe wide scale use of hybridization as a tool in human diagnostics.Among the more formidable problems are: 1) the inefficiency ofhybridization; 2) the low concentration of specific target sequences ina mixture of genomic DNA; and 3) the hybridization of only partiallycomplementary probes and targets.

With regard to efficiency, it is experimentally observed that only afraction of the possible number of probe-target complexes are formed ina hybridization reaction. This is particularly true with shortoligonucleotide probes (less than 100 bases in length). There are threefundamental causes: a) hybridization cannot occur because of secondaryand tertiary structure interactions; b) strands of DNA containing thetarget sequence have rehybridized (reannealed) to their complementarystrand; and c) some target molecules are prevented from hybridizationwhen they are used in hybridization formats that immobilize the targetnucleic acids to a solid surface.

Even where the sequence of a probe is completely complementary to thesequence of the target, i.e., the target's primary structure, the targetsequence must be made accessible to the probe via rearrangements ofhigher-order structure. These higher-order structural rearrangements mayconcern either the secondary structure or tertiary structure of themolecule. Secondary structure is determined by intramolecular bonding.In the case of DNA or RNA targets this consists of hybridization withina single, continuous strand of bases (as opposed to hybridizationbetween two different strands). Depending on the extent and position ofintramolecular bonding, the probe can be displaced from the targetsequence preventing hybridization.

Solution hybridization of oligonucleotide probes to denatureddouble-stranded DNA is further complicated by the fact that the longercomplementary target strands can renature or reanneal. Again, hybridizedprobe is displaced by this process. This results in a low yield ofhybridization (low “coverage”) relative to the starting concentrationsof probe and target.

With regard to low target sequence concentration, the DNA fragmentcontaining the target sequence is usually in relatively low abundance ingenomic DNA. This presents great technical difficulties; mostconventional methods that use oligonucleotide probes lack thesensitivity necessary to detect hybridization at such low levels.

One attempt at a solution to the target sequence concentration problemis the amplification of the detection signal. Most often this entailsplacing one or more labels on an oligonucleotide probe. In the case ofnon-radioactive labels, even the highest affinity reagents have beenfound to be unsuitable for the detection of single copy genes in genomicDNA with oligonucleotide probes. See Wallace et al., Biochimie 67:755(1985). In the case of radioactive oligonucleotide probes, onlyextremely high specific activities are found to show satisfactoryresults. See Studencki and Wallace, DNA 3:1 (1984) and Studencki et al.,Human Genetics 37:42 (1985).

With regard to complementarity, it is important for some diagnosticapplications to determine whether the hybridization represents completeor partial complementarity. For example, where it is desired to detectsimply the presence or absence of pathogen DNA (such as from a virus,bacterium, fungi, mycoplasma, protozoan) it is only important that thehybridization method ensures hybridization when the relevant sequence ispresent; conditions can be selected where both partially complementaryprobes and completely complementary probes will hybridize. Otherdiagnostic applications, however, may require that the hybridizationmethod distinguish between partial and complete complementarity. It maybe of interest to detect genetic polymorphisms. For example, humanhemoglobin is composed, in part, of four polypeptide chains. Two ofthese chains are identical chains of 141 amino acids (alpha chains) andtwo of these chains are identical chains of 146 amino acids (betachains). The gene encoding the beta chain is known to exhibitpolymorphism. The normal allele encodes a beta chain having glutamicacid at the sixth position. The mutant allele encodes a beta chainhaving valine at the sixth position. This difference in amino acids hasa profound (most profound when the individual is homozygous for themutant allele) physiological impact known clinically as sickle cellanemia. It is well known that the genetic basis of the amino acid changeinvolves a single base difference between the normal allele DNA sequenceand the mutant allele DNA sequence.

Unless combined with other techniques (such as restriction enzymeanalysis), methods that allow for the same level of hybridization in thecase of both partial as well as complete complementarity are typicallyunsuited for such applications; the probe will hybridize to both thenormal and variant target sequence. Hybridization, regardless of themethod used, requires some degree of complementarity between thesequence being assayed (the target sequence) and the fragment of DNAused to perform the test (the probe). (Of course, one can obtain bindingwithout any complementarity but this binding is nonspecific and to beavoided.)

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Certain bases not commonly found innatural nucleic acids may be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanineComplementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

Stability of a nucleic acid duplex is measured by the meltingtemperature, or “T_(m).” The T_(m) of a particular nucleic acid duplexunder specified conditions is the temperature at which on average halfof the base pairs have disassociated.

The term “probe” as used herein refers to a labeled oligonucleotidewhich forms a duplex structure with a sequence in another nucleic acid,due to complementarity of at least one sequence in the probe with asequence in the other nucleic acid.

The term “label” as used herein refers to any atom or molecule which canbe used to provide a detectable (preferably quantifiable) signal, andwhich can be attached to a nucleic acid or protein. Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like.

The term “cleavage structure” as used herein, refers to a region of asingle-stranded nucleic acid substrate containing secondary structure,said region being cleavable by a cleavage means, including but notlimited to an enzyme. The cleavage structure is a substrate for specificcleavage by said cleavage means in contrast to a nucleic acid moleculewhich is a substrate for non-specific cleavage by agents such asphosphodiesterases which cleave nucleic acid molecules without regard tosecondary structure (i.e., no folding of the substrate is required).

The term “cleavage means” as used herein refers to any means which iscapable of cleaving a cleavage structure, including but not limited toenzymes. The cleavage means may include native DNAPs having 5′ nucleaseactivity (e.g., Taq DNA polymerase, E. coli DNA polymerase I) and, morespecifically, modified DNAPs having 5′ nuclease but lacking syntheticactivity. The ability of 5′ nucleases to cleave naturally occurringstructures in nucleic acid templates (structure-specific cleavage) isuseful to detect internal sequence differences in nucleic acids withoutprior knowledge of the specific sequence of the nucleic acid. In thismanner, they are structure-specific enzymes. Structure-specific enzymesare enzymes which recognize specific secondary structures in a nucleicmolecule and cleave these structures. The site of cleavage may be oneither the 5′ or 3′ side of the cleavage structure; alternatively thesite of cleavage may be between the 5′ and 3′ side (i.e., within orinternal to) of the cleavage structure. The cleavage means of theinvention cleave a nucleic acid molecule in response to the formation ofcleavage structures; it is not necessary that the cleavage means cleavethe cleavage structure at any particular location within the cleavagestructure.

The cleavage means is not restricted to enzymes having 5′ nucleaseactivity. The cleavage means may include nuclease activity provided froma variety of sources including the enzyme CLEAVASE, Taq DNA polymerase,E. coli DNA polymerase I and eukaryotic structure-specificendonucleases, murine FEN-1 endonucleases (Harrington and Liener, (1994)Genes and Develop. 8:1344) and calf thymus 5′ to 3′ exonuclease(Murante, R. S., et al. (1994) J. Biol. Chem. 269:1191). In addition,enzymes having 3′ nuclease activity such as members of the family of DNArepair endonucleases (e.g., the RrpI enzyme from Drosophilamelanogaster, the yeast RAD1/RAD10 complex and E. coli Exo III), arealso suitable cleavage means for the practice of the methods of theinvention.

The term “cleavage products” as used herein, refers to productsgenerated by the reaction of a cleavage means with a cleavage structure(i.e., the treatment of a cleavage structure with a cleavage means).

The terms “nucleic acid substrate” and nucleic acid template” are usedherein interchangeably and refer to a nucleic acid molecule which whendenatured and allowed to renature (i.e., to fold upon itself by theformation of intra-strand hydrogen bonds), forms at least one cleavagestructure. The nucleic acid substrate may comprise single- ordouble-stranded DNA or RNA.

The term “substantially single-stranded” when used in reference to anucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

Nucleic acids form secondary structures which depend on base-pairing forstability. When single strands of nucleic acids (single-stranded DNA,denatured double-stranded DNA or RNA) with different sequences, evenclosely related ones, are allowed to fold on themselves, they assumecharacteristic secondary structures. At “elevated temperatures” theduplex regions of the structures are brought to the brink ofinstability, so that the effects of small changes in sequence aremaximized, and revealed as alterations in the cleavage pattern. In otherwords, “an elevated temperature” is a temperature at which a givenduplex region of the folded substrate molecule is near the temperatureat which that duplex melts. An alteration in the sequence of thesubstrate will then be likely to cause the destruction of a duplexregion(s) thereby generating a different cleavage pattern when acleavage agent which is dependent upon the recognition of structure isutilized in the reaction. While not being limited to any particulartheory, it is thought that individual molecules in the target (i.e., thesubstrate) population may each assume only one or a few of the potentialcleavage structures (i.e., duplexed regions), but when the sample isanalyzed as a whole, a composite pattern representing all cleavage sitesis detected. Many of the structures recognized as active cleavage sitesare likely to be only a few base-pairs long and would appear to beunstable when elevated temperatures used in the cleavage reaction.Nevertheless, transient formation of these structures allows recognitionand cleavage of these structures by said cleavage means. The formationor disruption of these structures in response to small sequence changesresults in changes in the patterns of cleavage. Temperatures in therange of 40-85° C., with the range of 55-85° C. being particularlypreferred, are suitable elevated temperatures for the practice of themethod of the invention.

The term “sequence variation” as used herein refers to differences innucleic acid sequence between two nucleic acid templates. For example, awild-type structural gene and a mutant form of this wild-type structuralgene may vary in sequence by the presence of single base substitutionsand/or deletions or insertions of one or more nucleotides. These twoforms of the structural gene are said to vary in sequence from oneanother. A second mutant form of the structural gene may exits. Thissecond mutant form is said to vary in sequence from both the wild-typegene and the first mutant form of the gene. It is noted, however, thatthe invention does not require that a comparison be made between one ormore forms of a gene to detect sequence variations. Because the methodof the invention generates a characteristic and reproducible pattern ofcleavage products for a given nucleic acid substrate, a characteristic“fingerprint” may be obtained from any nucleic substrate withoutreference to a wild-type or other control. The invention contemplatesthe use of the method for both “fingerprinting” nucleic acids withoutreference to a control and identification of mutant forms of a substratenucleic acid by comparison of the mutant form of the substrate with awild-type or known mutant control.

The term “liberating” as used herein refers to the release of a nucleicacid fragment from a larger nucleic acid fragment, such as anoligonucleotide, by the action of a 5′ nuclease such that the releasedfragment is no longer covalently attached to the remainder of theoligonucleotide.

The term “substrate strand” as used herein, means that strand of nucleicacid in a cleavage structure in which the cleavage mediated by the 5′nuclease activity occurs.

The term “template strand” as used herein, means that strand of nucleicacid in a cleavage structure which is at least partially complementaryto the substrate strand and which anneals to the substrate strand toform the cleavage structure.

The term “K_(m)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction.

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides such as 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogsand comprise modified forms of deoxyribonucleotides as well asribonucleotides. As used herein the term “nucleotide analog” when usedin reference to substrates present in a PCR mixture refers to the use ofnucleotides other than dATP, dGTP, dCTP and dTTP; thus, the use of dUTP(a naturally occurring dNTP) in a PCR would comprise the use of anucleotide analog in the PCR. A PCR product generated using dUTP,7-deaza-dATP, 7-deaza-dGTP or any other nucleotide analog in thereaction mixture is said to contain nucleotide analogs.

“Oligonucleotide primers matching or complementary to a gene sequence”refers to oligonucleotide primers capable of facilitating thetemplate-dependent synthesis of single or double-stranded nucleic acids.Oligonucleotide primers matching or complementary to a gene sequence maybe used in PCRs, RT-PCRs and the like.

A “consensus gene sequence” refers to a gene sequence which is derivedby comparison of two or more gene sequences and which describes thenucleotides most often present in a given segment of the genes; theconsensus sequence is the canonical sequence.

The term “polymorphic locus” is a locus present in a population whichshows variation between members of the population (i.e., the most commonallele has a frequency of less than 0.95). In contrast, a “monomorphiclocus” is a genetic locus at little or no variations seen betweenmembers of the population (generally taken to be a locus at which themost common allele exceeds a frequency of 0.95 in the gene pool of thepopulation).

The term “microorganism” as used herein means an organism too small tobe observed with the unaided eye and includes, but is not limited tobacteria, virus, protozoans, fungi, and ciliates.

The term “microbial gene sequences” refers to gene sequences derivedfrom a microorganism.

The term “bacteria” refers to any bacterial species includingeubacterial and archaebacterial species.

The term “virus” refers to obligate, ultramicroscopic, intracellularparasites incapable of autonomous replication (i.e., replicationrequires the use of the host cell's machinery).

The term “multi-drug resistant” or multiple-drug resistant” refers to amicroorganism which is resistant to more than one of the antibiotics orantimicrobial agents used in the treatment of said microorganism.

DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for treatingnucleic acid, and in particular, methods and compositions for detectionand characterization of nucleic acid sequences and sequence changes.

The present invention relates to means for cleaving a nucleic acidcleavage structure in a site-specific manner. In particular, the presentinvention relates to a cleaving enzyme having 5′ nuclease activitywithout interfering nucleic acid synthetic ability.

This invention provides 5′ nucleases derived from thermostable DNApolymerases which exhibit altered DNA synthetic activity from that ofnative thermostable DNA polymerases. The 5′ nuclease activity of thepolymerase is retained while the synthetic activity is reduced orabsent. Such 5′ nucleases are capable of catalyzing thestructure-specific cleavage of nucleic acids in the absence ofinterfering synthetic activity. The lack of synthetic activity during acleavage reaction results in nucleic acid cleavage products of uniformsize.

The novel properties of the polymerases of the invention form the basisof a method of detecting specific nucleic acid sequences. This methodrelies upon the amplification of the detection molecule rather than uponthe amplification of the target sequence itself as do existing methodsof detecting specific target sequences.

DNA polymerases (DNAPs), such as those isolated from E. coli or fromthermophilic bacteria of the genus Thermus, are enzymes that synthesizenew DNA strands. Several of the known DNAPs contain associated nucleaseactivities in addition to the synthetic activity of the enzyme.

Some DNAPs are known to remove nucleotides from the 5′ and 3′ ends ofDNA chains (Kornberg, DNA Replication, W.H. Freeman and Co., SanFrancisco, pp. 127-139 (1980)). These nuclease activities are usuallyreferred to as 5′ exonuclease and 3′ exonuclease activities,respectively. For example, the 5′ exonuclease activity located in theN-terminal domain of several DNAPs participates in the removal of RNAprimers during lagging strand synthesis during DNA replication and theremoval of damaged nucleotides during repair. Some DNAPs, such as the E.coli DNA polymerase (DNAPEc1), also have a 3′ exonuclease activityresponsible for proof-reading during DNA synthesis (Kornberg, supra).

A DNAP isolated from Thermus aquaticus, termed Taq DNA polymerase(DNAPTaq), has a 5′ exonuclease activity, but lacks a functional 3′exonucleolytic domain (Tindall and Kunkell, Biochem. 27:6008 (1988)).Derivatives of DNAPEc1 and DNAPTaq, respectively called the Klenow andStoffel fragments, lack 5′ exonuclease domains as a result of enzymaticor genetic manipulations (Brutlag et al., Biochem. Biophys. Res. Commun.37:982 (1969); Erlich et al., Science 252:1643 (1991); Setlow andKornberg, J. Biol. Chem. 247:232 (1972)).

The 5′ exonuclease activity of DNAPTaq was reported to requireconcurrent synthesis (Gelfand, PCR Technology—Principles andApplications for DNA Amplification (H. A. Erlich, Ed.), Stockton Press,New York, p. 19 (1989)). Although mononucleotides predominate among thedigestion products of the 5′ exonucleases of DNAPTaq and DNAPEc1, shortoligonucleotides (≦12 nucleotides) can also be observed implying thatthese so-called 5′ exonucleases can function endonucleolytically(Setlow, supra; Holland et al., Proc. Natl. Acad. Sci. USA 88:7276(1991)).

In WO 92/06200, Gelfand et al. show that the preferred substrate of the5′ exonuclease activity of the thermostable DNA polymerases is displacedsingle-stranded DNA. Hydrolysis of the phosphodiester bond occursbetween the displaced single-stranded DNA and the double-helical DNAwith the preferred exonuclease cleavage site being a phosphodiester bondin the double helical region. Thus, the 5′ exonuclease activity usuallyassociated with DNAPs is a structure-dependent single-strandedendonuclease and is more properly referred to as a 5′ nuclease.Exonucleases are enzymes which cleave nucleotide molecules from the endsof the nucleic acid molecule. Endonucleases, on the other hand, areenzymes which cleave the nucleic acid molecule at internal rather thanterminal sites. The nuclease activity associated with some thermostableDNA polymerases cleaves endonucleolytically but this cleavage requirescontact with the 5′ end of the molecule being cleaved. Therefore, thesenucleases are referred to as 5′ nucleases.

When a 5′ nuclease activity is associated with a eubacterial Type A DNApolymerase, it is found in the one-third N-terminal region of theprotein as an independent functional domain. The C-terminal two-thirdsof the molecule constitute the polymerization domain which isresponsible for the synthesis of DNA. Some Type A DNA polymerases alsohave a 3′ exonuclease activity associated with the two-third C-terminalregion of the molecule.

The 5′ exonuclease activity and the polymerization activity of DNAPshave been separated by proteolytic cleavage or genetic manipulation ofthe polymerase molecule. To date thermostable DNAPs have been modifiedto remove or reduce the amount of 5′ nuclease activity while leaving thepolymerase activity intact.

The Klenow or large proteolytic cleavage fragment of DNAPEc1 containsthe polymerase and 3′ exonuclease activity but lacks the 5′ nucleaseactivity. The Stoffel fragment of DNAPTaq (DNAPStf) lacks the 5′nuclease activity due to a genetic manipulation which deleted theN-terminal 289 amino acids of the polymerase molecule (Erlich et al.,Science 252:1643 (1991)). WO 92/06200 describes a thermostable DNAP withan altered level of 5′ to 3′ exonuclease. U.S. Pat. No. 5,108,892describes a Thermus aquaticus DNAP without a 5′ to 3′ exonuclease.However, the art of molecular biology lacks a thermostable DNApolymerase with a lessened amount of synthetic activity.

The present invention provides 5′ nucleases derived from thermostableType A DNA polymerases that retain 5′ nuclease activity but have reducedor absent synthetic activity. The ability to uncouple the syntheticactivity of the enzyme from the 5′ nuclease activity proves that the 5′nuclease activity does not require concurrent DNA synthesis as waspreviously reported (Gelfand, PCR Technology, supra).

The description of the invention is divided into: I. Detection ofSpecific Nucleic Acid Sequences Using 5′ Nucleases; II. Generation of 5′Nucleases Derived From Thermostable DNA Polymerases; III. TherapeuticUses of 5′ Nucleases; IV. Detection of Antigenic or Nucleic Acid Targetsby a Dual Capture Assay; and V. CLEAVASE Fragment Length Polymorphismfor the Detection of Secondary Structure and VI. Detection of Mutationsin the p53 Tumor Suppressor Gene Using the CFLP Method.

I. Detection of Specific Nucleic Acid Sequences Using 5′ Nucleases

The 5′ nucleases of the invention form the basis of a novel detectionassay for the identification of specific nucleic acid sequences. Thisdetection system identifies the presence of specific nucleic acidsequences by requiring the annealing of two oligonucleotide probes totwo portions of the target sequence. As used herein, the term “targetsequence” or “target nucleic acid sequence” refers to a specific nucleicacid sequence within a polynucleotide sequence, such as genomic DNA orRNA, which is to be either detected or cleaved or both.

FIG. 1A provides a schematic of one embodiment of the detection methodof the present invention. The target sequence is recognized by twodistinct oligonucleotides in the triggering or trigger reaction. It ispreferred that one of these oligonucleotides is provided on a solidsupport. The other can be provided free. In FIG. 1A the free oligo isindicated as a “primer” and the other oligo is shown attached to a beaddesignated as type 1. The target nucleic acid aligns the twooligonucleotides for specific cleavage of the 5′ arm (of the oligo onbead 1) by the DNAPs of the present invention (not shown in FIG. 1A).

The site of cleavage (indicated by a large solid arrowhead) iscontrolled by the distance between the 3′ end of the “primer” and thedownstream fork of the oligo on bead 1. The latter is designed with anuncleavable region (indicated by the striping). In this manner neitheroligonucleotide is subject to cleavage when misaligned or whenunattached to target nucleic acid.

Successful cleavage releases a single copy of what is referred to as thealpha signal oligo. This oligo may contain a detectable moiety (e.g.,fluorescein). On the other hand, it may be unlabelled.

In one embodiment of the detection method, two more oligonucleotides areprovided on solid supports. The oligonucleotide shown in FIG. 1A on bead2 has a region that is complementary to the alpha signal oligo(indicated as alpha prime) allowing for hybridization. This structurecan be cleaved by the DNAPs of the present invention to release the betasignal oligo. The beta signal oligo can then hybridize to type 3 beadshaving an oligo with a complementary region (indicated as beta prime).Again, this structure can be cleaved by the DNAPs of the presentinvention to release a new alpha oligo.

At this point, the amplification has been linear. To increase the powerof the method, it is desired that the alpha signal oligo hybridized tobead type 2 be liberated after release of the beta oligo so that it maygo on to hybridize with other oligos on type 2 beads. Similarly, afterrelease of an alpha oligo from type 3 beads, it is desired that the betaoligo be liberated.

The liberation of “captured” signal oligos can be achieved in a numberof ways. First, it has been found that the DNAPs of the presentinvention have a true 5′ exonuclease capable of “nibbling” the 5′ end ofthe alpha (and beta) prime oligo (discussed below in more detail). Thus,under appropriate conditions, the hybridization is destabilized bynibbling of the DNAP. Second, the alpha-alpha prime (as well as thebeta—beta prime) complex can be destabilized by heat (e.g., thermalcycling).

With the liberation of signal oligos by such techniques, each cleavageresults in a doubling of the number of signal oligos. In this manner,detectable signal can quickly be achieved.

FIG. 1B provides a schematic of a second embodiment of the detectionmethod of the present invention. Again, the target sequence isrecognized by two distinct oligonucleotides in the triggering or triggerreaction and the target nucleic acid aligns the two oligonucleotides forspecific cleavage of the 5′ arm by the DNAPs of the present invention(not shown in FIG. 1B). The first oligo is completely complementary to aportion of the target sequence. The second oligonucleotide is partiallycomplementary to the target sequence; the 3′ end of the secondoligonucleotide is fully complementary to the target sequence while the5′ end is non-complementary and forms a single-stranded arm. Thenon-complementary end of the second oligonucleotide may be a genericsequence which can be used with a set of standard hairpin structures(described below). The detection of different target sequences wouldrequire unique portions of two oligonucleotides: the entire firstoligonucleotide and the 3′ end of the second oligonucleotide. The 5′ armof the second oligonucleotide can be invariant or generic in sequence.

The annealing of the first and second oligonucleotides near one anotheralong the target sequence forms a forked cleavage structure which is asubstrate for the 5′ nuclease of DNA polymerases. The approximatelocation of the cleavage site is again indicated by the large solidarrowhead in FIG. 1B.

The 5′ nucleases of the invention are capable of cleaving this structurebut are not capable of polymerizing the extension of the 3′ end of thefirst oligonucleotide. The lack of polymerization activity isadvantageous as extension of the first oligonucleotide results indisplacement of the annealed region of the second oligonucleotide andresults in moving the site of cleavage along the second oligonucleotide.If polymerization is allowed to occur to any significant amount,multiple lengths of cleavage product will be generated. A singlecleavage product of uniform length is desirable as this cleavage productinitiates the detection reaction.

The trigger reaction may be run under conditions that allow forthermocycling. Thermocycling of the reaction allows for a logarithmicincrease in the amount of the trigger oligonucleotide released in thereaction.

The second part of the detection method allows the annealing of thefragment of the second oligonucleotide liberated by the cleavage of thefirst cleavage structure formed in the triggering reaction (called thethird or trigger oligonucleotide) to a first hairpin structure. Thisfirst hairpin structure has a single-stranded 5′ arm and asingle-stranded 3′ arm. The third oligonucleotide triggers the cleavageof this first hairpin structure by annealing to the 3′ arm of thehairpin thereby forming a substrate for cleavage by the 5′ nuclease ofthe present invention. The cleavage of this first hairpin structuregenerates two reaction products: 1) the cleaved 5′ arm of the hairpincalled the fourth oligonucleotide, and 2) the cleaved hairpin structurewhich now lacks the 5′ arm and is smaller in size than the uncleavedhairpin. This cleaved first hairpin may be used as a detection moleculeto indicate that cleavage directed by the trigger or thirdoligonucleotide occurred. Thus, this indicates that the first twooligonucleotides found and annealed to the target sequence therebyindicating the presence of the target sequence in the sample.

The detection products are amplified by having the fourtholigonucleotide anneal to a second hairpin structure. This hairpinstructure has a 5′ single-stranded arm and a 3′ single-stranded arm. Thefourth oligonucleotide generated by cleavage of the first hairpinstructure anneals to the 3′ arm of the second hairpin structure therebycreating a third cleavage structure recognized by the 5′ nuclease. Thecleavage of this second hairpin structure also generates two reactionproducts: 1) the cleaved 5′ arm of the hairpin called the fiftholigonucleotide which is similar or identical in sequence to the thirdnucleotide, and 2) the cleaved second hairpin structure which now lacksthe 5′ arm and is smaller in size than the uncleaved hairpin. Thiscleaved second hairpin may be as a detection molecule and amplifies thesignal generated by the cleavage of the first hairpin structure.Simultaneously with the annealing of the forth oligonucleotide, thethird oligonucleotide is dissociated from the cleaved first hairpinmolecule so that it is free to anneal to a new copy of the first hairpinstructure. The disassociation of the oligonucleotides from the hairpinstructures may be accomplished by heating or other means suitable todisrupt base-pairing interactions.

Further amplification of the detection signal is achieved by annealingthe fifth oligonucleotide (similar or identical in sequence to the thirdoligonucleotide) to another molecule of the first hairpin structure.Cleavage is then performed and the oligonucleotide that is liberatedthen is annealed to another molecule of the second hairpin structure.Successive rounds of annealing and cleavage of the first and secondhairpin structures, provided in excess, are performed to generate asufficient amount of cleaved hairpin products to be detected. Thetemperature of the detection reaction is cycled just below and justabove the annealing temperature for the oligonucleotides used to directcleavage of the hairpin structures, generally about 55° C. to 70° C. Thenumber of cleavages will double in each cycle until the amount ofhairpin structures remaining is below the K_(m) for the hairpinstructures. This point is reached when the hairpin structures aresubstantially used up. When the detection reaction is to be used in aquantitative manner, the cycling reactions are stopped before theaccumulation of the cleaved hairpin detection products reach a plateau.

Detection of the cleaved hairpin structures may be achieved in severalways. In one embodiment detection is achieved by separation on agaroseor polyacrylamide gels followed by staining with ethidium bromide. Inanother embodiment, detection is achieved by separation of the cleavedand uncleaved hairpin structures on a gel followed by autoradiographywhen the hairpin structures are first labelled with a radioactive probeand separation on chromatography columns using HPLC or FPLC followed bydetection of the differently sized fragments by absorption at OD₂₆₀.Other means of detection include detection of changes in fluorescencepolarization when the single-stranded 5′ arm is released by cleavage,the increase in fluorescence of an intercalating fluorescent indicatoras the amount of primers annealed to 3′ arms of the hairpin structuresincreases. The formation of increasing amounts of duplex DNA (betweenthe primer and the 3′ arm of the hairpin) occurs if successive rounds ofcleavage occur.

The hairpin structures may be attached to a solid support, such as anagarose, styrene or magnetic bead, via the 3′ end of the hairpin. Aspacer molecule may be placed between the 3′ end of the hairpin and thebead, if so desired. The advantage of attaching the hairpin structuresto a solid support is that this prevents the hybridization of the twohairpin structures to one another over regions which are complementary.If the hairpin structures anneal to one another, this would reduce theamount of hairpins available for hybridization to the primers releasedduring the cleavage reactions. If the hairpin structures are attached toa solid support, then additional methods of detection of the products ofthe cleavage reaction may be employed. These methods include, but arenot limited to, the measurement of the released single-stranded 5′ armwhen the 5′ arm contains a label at the 5′ terminus. This label may beradioactive, fluorescent, biotinylated, etc. If the hairpin structure isnot cleaved, the 5′ label will remain attached to the solid support. Ifcleavage occurs, the 5′ label will be released from the solid support.

The 3′ end of the hairpin molecule may be blocked through the use ofdideoxynucleotides. A 3′ terminus containing a dideoxynucleotide isunavailable to participate in reactions with certain DNA modifyingenzymes, such as terminal transferase. Cleavage of the hairpin having a3′ terminal dideoxynucleotide generates a new, unblocked 3′ terminus atthe site of cleavage. This new 3′ end has a free hydroxyl group whichcan interact with terminal transferase thus providing another means ofdetecting the cleavage products.

The hairpin structures are designed so that their self-complementaryregions are very short (generally in the range of 3-8 base pairs). Thus,the hairpin structures are not stable at the high temperatures at whichthis reaction is performed (generally in the range of 50-75° C.) unlessthe hairpin is stabilized by the presence of the annealedoligonucleotide on the 3′ arm of the hairpin. This instability preventsthe polymerase from cleaving the hairpin structure in the absence of anassociated primer thereby preventing false positive results due tonon-oligonucleotide directed cleavage.

As discussed above, the use of the 5′ nucleases of the invention whichhave reduced polymerization activity is advantageous in this method ofdetecting specific nucleic acid sequences. Significant amounts ofpolymerization during the cleavage reaction would cause shifting of thesite of cleavage in unpredictable ways resulting in the production of aseries of cleaved hairpin structures of various sizes rather than asingle easily quantifiable product. Additionally, the primers used inone round of cleavage could, if elongated, become unusable for the nextcycle, by either forming an incorrect structure or by being too long tomelt off under moderate temperature cycling conditions. In a pristinesystem (i.e., lacking the presence of dNTPs), one could use theunmodified polymerase, but the presence of nucleotides (dNTPs) candecrease the per cycle efficiency enough to give a false negativeresult. When a crude extract (genomic DNA preparations, crude celllysates, etc.) is employed or where a sample of DNA from a PCR reaction,or any other sample that might be contaminated with dNTPs, the 5′nucleases of the present invention that were derived from thermostablepolymerases are particularly useful.

II. Generation of 5′ Nucleases from Thermostable DNA Polymerases

The genes encoding Type A DNA polymerases share about 85% homology toeach other on the DNA sequence level. Preferred examples of thermostablepolymerases include those isolated from Thermus aquaticus, Thermusflavus, and Thermus thermophilus. However, other thermostable Type Apolymerases which have 5′ nuclease activity are also suitable. FIGS. 2and 3 compare the nucleotide and amino acid sequences of the three abovementioned polymerases. In FIGS. 2 and 3, the consensus or majoritysequence derived from a comparison of the nucleotide (FIG. 2) or aminoacid (FIG. 3) sequence of the three thermostable DNA polymerases isshown on the top line. A dot appears in the sequences of each of thesethree polymerases whenever an amino acid residue in a given sequence isidentical to that contained in the consensus amino acid sequence. Dashesare used to introduce gaps in order to maximize alignment between thedisplayed sequences. When no consensus nucleotide or amino acid ispresent at a given position, an “X” is placed in the consensus sequence.SEQ ID NOS:1-3 display the nucleotide sequences and SEQ ID NOS:4-6display the amino acid sequences of the three wild-type polymerases. SEQID NO:1 corresponds to the nucleic acid sequence of the wild typeThermus aquaticus DNA polymerase gene isolated from the YT-1 strain(Lawyer et al., J. Biol. Chem. 264:6427 (1989)). SEQ ID NO:2 correspondsto the nucleic acid sequence of the wild type Thermus flavus DNApolymerase gene (Akhmetzjanov and Vakhitov, Nucl. Acids Res. 20:5839(1992)). SEQ ID NO:3 corresponds to the nucleic acid sequence of thewild type Thermus thermophilus DNA polymerase gene (Gelfand et al., WO91/09950 (1991)). SEQ ID NOS:7-8 depict the consensus nucleotide andamino acid sequences, respectively for the above three DNAPs (also shownon the top row in FIGS. 2 and 3).

The 5′ nucleases of the invention derived from thermostable polymeraseshave reduced synthetic ability, but retain substantially the same 5′exonuclease activity as the native DNA polymerase. The term“substantially the same 5′ nuclease activity” as used herein means thatthe 5′ nuclease activity of the modified enzyme retains the ability tofunction as a structure-dependent single-stranded endonuclease but notnecessarily at the same rate of cleavage as compared to the unmodifiedenzyme. Type A DNA polymerases may also be modified so as to produce anenzyme which has increases 5′ nuclease activity while having a reducedlevel of synthetic activity. Modified enzymes having reduced syntheticactivity and increased 5′ nuclease activity are also envisioned by thepresent invention.

By the term “reduced synthetic activity” as used herein it is meant thatthe modified enzyme has less than the level of synthetic activity foundin the unmodified or “native” enzyme. The modified enzyme may have nosynthetic activity remaining or may have that level of syntheticactivity that will not interfere with the use of the modified enzyme inthe detection assay described below. The 5′ nucleases of the presentinvention are advantageous in situations where the cleavage activity ofthe polymerase is desired, but the synthetic ability is not (such as inthe detection assay of the invention).

As noted above, it is not intended that the invention be limited by thenature of the alteration necessary to render the polymerase synthesisdeficient. The present invention contemplates a variety of methods,including but not limited to: 1) proteolysis; 2) recombinant constructs(including mutants); and 3) physical and/or chemical modification and/orinhibition.

1. Proteolysis

Thermostable DNA polymerases having a reduced level of syntheticactivity are produced by physically cleaving the unmodified enzyme withproteolytic enzymes to produce fragments of the enzyme that aredeficient in synthetic activity but retain 5′ nuclease activity.Following proteolytic digestion, the resulting fragments are separatedby standard chromatographic techniques and assayed for the ability tosynthesize DNA and to act as a 5′ nuclease. The assays to determinesynthetic activity and 5′ nuclease activity are described below.

2. Recombinant Constructs

The examples below describe a preferred method for creating a constructencoding a 5′ nuclease derived from a thermostable DNA polymerase. Asthe Type A DNA polymerases are similar in DNA sequence, the cloningstrategies employed for the Thermus aquaticus and flavus polymerases areapplicable to other thermostable Type A polymerases. In general, athermostable DNA polymerase is cloned by isolating genomic DNA usingmolecular biological methods from a bacteria containing a thermostableType A DNA polymerase. This genomic DNA is exposed to primers which arecapable of amplifying the polymerase gene by PCR.

This amplified polymerase sequence is then subjected to standarddeletion processes to delete the polymerase portion of the gene.Suitable deletion processes are described below in the examples.

The example below discusses the strategy used to determine whichportions of the DNAPTaq polymerase domain could be removed withouteliminating the 5′ nuclease activity. Deletion of amino acids from theprotein can be done either by deletion of the encoding genetic material,or by introduction of a translational stop codon by mutation or frameshift. In addition, proteolytic treatment of the protein molecule can beperformed to remove segments of the protein.

In the examples below, specific alterations of the Taq gene were: adeletion between nucleotides 1601 and 2502 (the end of the codingregion), a 4 nucleotide insertion at position 2043, and deletionsbetween nucleotides 1614 and 1848 and between nucleotides 875 and 1778(numbering is as in SEQ ID NO:1). These modified sequences are describedbelow in the examples and at SEQ ID NOS:9-12.

Those skilled in the art understand that single base pair changes can beinnocuous in terms of enzyme structure and function. Similarly, smalladditions and deletions can be present without substantially changingthe exonuclease or polymerase function of these enzymes.

Other deletions are also suitable to create the 5′ nucleases of thepresent invention. It is preferable that the deletion decrease thepolymerase activity of the 5′ nucleases to a level at which syntheticactivity will not interfere with the use of the 5′ nuclease in thedetection assay of the invention. Most preferably, the synthetic abilityis absent. Modified polymerases are tested for the presence of syntheticand 5′ nuclease activity as in assays described below. Thoughtfulconsideration of these assays allows for the screening of candidateenzymes whose structure is heretofore as yet unknown. In other words,construct “X” can be evaluated according to the protocol described belowto determine whether it is a member of the genus of 5′ nucleases of thepresent invention as defined functionally, rather than structurally.

In the example below, the PCR product of the amplified Thermus aquaticusgenomic DNA did not have the identical nucleotide structure of thenative genomic DNA and did not have the same synthetic ability of theoriginal clone. Base pair changes which result due to the infidelity ofDNAPTaq during PCR amplification of a polymerase gene are also a methodby which the synthetic ability of a polymerase gene may be inactivated.The examples below and FIGS. 4A and 5A indicate regions in the nativeThermus aquaticus and flavus DNA polymerases likely to be important forsynthetic ability. There are other base pair changes and substitutionsthat will likely also inactivate the polymerase.

It is not necessary, however, that one start out the process ofproducing a 5′ nuclease from a DNA polymerase with such a mutatedamplified product. This is the method by which the examples below wereperformed to generate the synthesis-deficient DNAPTaq mutants, but it isunderstood by those skilled in the art that a wild-type DNA polymerasesequence may be used as the starting material for the introduction ofdeletions, insertion and substitutions to produce a 5′ nuclease. Forexample, to generate the synthesis-deficient DNAPTf1 mutant, the primerslisted in SEQ ID NOS:13-14 were used to amplify the wild type DNApolymerase gene from Thermus flavus strain AT-62. The amplifiedpolymerase gene was then subjected to restriction enzyme digestion todelete a large portion of the domain encoding the synthetic activity.

The present invention contemplates that the nucleic acid construct ofthe present invention be capable of expression in a suitable host. Thosein the art know methods for attaching various promoters and 3′ sequencesto a gene structure to achieve efficient expression. The examples belowdisclose two suitable vectors and six suitable vector constructs. Ofcourse, there are other promoter/vector combinations that would besuitable. It is not necessary that a host organism be used for theexpression of the nucleic acid constructs of the invention. For example,expression of the protein encoded by a nucleic acid construct may beachieved through the use of a cell-free in vitrotranscription/translation system. An example of such a cell-free systemis the commercially available TNT Coupled Reticulocyte Lysate System(Promega Corporation, Madison, Wis.).

Once a suitable nucleic acid construct has been made, the 5′ nucleasemay be produced from the construct. The examples below and standardmolecular biological teachings enable one to manipulate the construct bydifferent suitable methods.

Once the 5′ nuclease has been expressed, the polymerase is tested forboth synthetic and nuclease activity as described below.

3. Physical and/or Chemical Modification and/or Inhibition

The synthetic activity of a thermostable DNA polymerase may be reducedby chemical and/or physical means. In one embodiment, the cleavagereaction catalyzed by the 5′ nuclease activity of the polymerase is rununder conditions which preferentially inhibit the synthetic activity ofthe polymerase. The level of synthetic activity need only be reduced tothat level of activity which does not interfere with cleavage reactionsrequiring no significant synthetic activity.

As shown in the examples below, concentrations of Mg⁺⁺ greater than 5 mMinhibit the polymerization activity of the native DNAPTaq. The abilityof the 5′ nuclease to function under conditions where synthetic activityis inhibited is tested by running the assays for synthetic and 5′nuclease activity, described below, in the presence of a range of Mg⁺⁺concentrations (5 to 10 mM). The effect of a given concentration of Mg⁺⁺is determined by quantitation of the amount of synthesis and cleavage inthe test reaction as compared to the standard reaction for each assay.

The inhibitory effect of other ions, polyamines, denaturants, such asurea, formamide, dimethylsulfoxide, glycerol and non-ionic detergents(TRITON X-100 and TWEEN-20), nucleic acid binding chemicals such as,actinomycin D, ethidium bromide and psoralens, are tested by theiraddition to the standard reaction buffers for the synthesis and 5′nuclease assays. Those compounds having a preferential inhibitory effecton the synthetic activity of a thermostable polymerase are then used tocreate reaction conditions under which 5′ nuclease activity (cleavage)is retained while synthetic activity is reduced or eliminated.

Physical means may be used to preferentially inhibit the syntheticactivity of a polymerase. For example, the synthetic activity ofthermostable polymerases is destroyed by exposure of the polymerase toextreme heat (typically 96 to 100° C.) for extended periods of time(greater than or equal to 20 minutes). While these are minor differenceswith respect to the specific heat tolerance for each of the enzymes,these are readily determined. Polymerases are treated with heat forvarious periods of time and the effect of the heat treatment upon thesynthetic and 5′ nuclease activities is determined.

III. Therapeutic Utility of 5′ Nucleases

The 5′ nucleases of the invention have not only the diagnostic utilitydiscussed above, but additionally have therapeutic utility for thecleavage and inactivation of specific mRNAs inside infected cells. ThemRNAs of pathogenic agents, such as viruses, bacteria, are targeted forcleavage by a synthesis-deficient DNA polymerase by the introduction ofa oligonucleotide complementary to a given mRNA produced by thepathogenic agent into the infected cell along with thesynthesis-deficient polymerase. Any pathogenic agent may be targeted bythis method provided the nucleotide sequence information is available sothat an appropriate oligonucleotide may be synthesized. The syntheticoligonucleotide anneals to the complementary mRNA thereby forming acleavage structure recognized by the modified enzyme. The ability of the5′ nuclease activity of thermostable DNA polymerases to cleave RNA-DNAhybrids is shown herein in Example 1D.

Liposomes provide a convenient delivery system. The syntheticoligonucleotide may be conjugated or bound to the nuclease to allow forco-delivery of these molecules. Additional delivery systems may beemployed.

Inactivation of pathogenic mRNAs has been described using antisense generegulation and using ribozymes (Rossi, U.S. Pat. No. 5,144,019, herebyincorporated by reference). Both of these methodologies havelimitations.

The use of antisense RNA to impair gene expression requiresstoichiometric and therefore, large molar excesses of anti-sense RNArelative to the pathogenic RNA to be effective. Ribozyme therapy, on theother hand, is catalytic and therefore lacks the problem of the need fora large molar excess of the therapeutic compound found with antisensemethods. However, ribozyme cleavage of a given RNA requires the presenceof highly conserved sequences to form the catalytically active cleavagestructure. This requires that the target pathogenic mRNA contain theconserved sequences (GAAAC (X)_(n) GU) thereby limiting the number ofpathogenic mRNAs that can be cleaved by this method. In contrast, thecatalytic cleavage of RNA by the use of a DNA oligonucleotide and a 5′nuclease is dependent upon structure only; thus, virtually anypathogenic RNA sequence can be used to design an appropriate cleavagestructure.

IV. Detection of Antigenic or Nucleic Acid Targets by a Dual CaptureAssay

The ability to generate 5′ nucleases from thermostable DNA polymerasesprovides the basis for a novel means of detecting the presence ofantigenic or nucleic acid targets. In this dual capture assay, thepolymerase domains encoding the synthetic activity and the nucleaseactivity are covalently attached to two separate and distinct antibodiesor oligonucleotides. When both the synthetic and the nuclease domainsare present in the same reaction and dATP, dTTP and a small amount ofpoly d(A-T) are provided, an enormous amount of poly d(A-T) is produced.The large amounts of poly d(A-T) are produced as a result of the abilityof the 5′ nuclease to cleave newly made poly d(A-T) to generate primersthat are, in turn, used by the synthetic domain to catalyze theproduction of even more poly d(A-T). The 5′ nuclease is able to cleavepoly d(A-T) because poly d(A-T) is self-complementary and easily formsalternate structures at elevated temperatures. These structures arerecognized by the 5′ nuclease and are then cleaved to generate moreprimer for the synthesis reaction.

The following is an example of the dual capture assay to detect anantigen(s): A sample to be analyzed for a given antigen(s) is provided.This sample may comprise a mixture of cells; for example, cells infectedwith viruses display virally-encoded antigens on their surface. If theantigen(s) to be detected are present in solution, they are firstattached to a solid support such as the wall of a microtiter dish or toa bead using conventional methodologies. The sample is then mixedwith 1) the synthetic domain of a thermostable DNA polymerase conjugatedto an antibody which recognizes either a first antigen or a firstepitope on an antigen, and 2) the 5′ nuclease domain of a thermostableDNA polymerase conjugated to a second antibody which recognizes either asecond, distinct antigen or a second epitope on the same antigen asrecognized by the antibody conjugated to the synthetic domain. Followingan appropriate period to allow the interaction of the antibodies withtheir cognate antigens (conditions will vary depending upon theantibodies used; appropriate conditions are well known in the art), thesample is then washed to remove unbound antibody-enzyme domaincomplexes. dATP, dTTP and a small amount of poly d(A-T) is then added tothe washed sample and the sample is incubated at elevated temperatures(generally in the range of 60-80° C. and more preferably, 70-75° C.) topermit the thermostable synthetic and 5′ nuclease domains to function.If the sample contains the antigen(s) recognized by both separatelyconjugated domains of the polymerase, then an exponential increase inpoly d(A-T) production occurs. If only the antibody conjugated to thesynthetic domain of the polymerase is present in the sample such that no5′ nuclease domain is present in the washed sample, then only anarithmetic increase in poly d(A-T) is possible. The reaction conditionsmay be controlled in such a way so that an arithmetic increase in polyd(A-T) is below the threshold of detection. This may be accomplished bycontrolling the length of time the reaction is allowed to proceed or byadding so little poly d(A-T) to act as template that in the absence ofnuclease activity to generate new poly d(A-T) primers very little polyd(A-T) is synthesized.

It is not necessary for both domains of the enzyme to be conjugated toan antibody. One can provide the synthetic domain conjugated to anantibody and provide the 5′ nuclease domain in solution or vice versa.In such a case the conjugated antibody-enzyme domain is added to thesample, incubated, then washed. dATP, dTTP, poly d(A-T) and theremaining enzyme domain in solution is then added.

Additionally, the two enzyme domains may be conjugated tooligonucleotides such that target nucleic acid sequences can bedetected. The oligonucleotides conjugated to the two different enzymedomains may recognize different regions on the same target nucleic acidstrand or may recognize two unrelated target nucleic acids.

The production of poly d(A-T) may be detected in many ways including: 1)use of a radioactive label on either the dATP or dTTP supplied for thesynthesis of the poly d(A-T), followed by size separation of thereaction products and autoradiography; 2) use of a fluorescent probe onthe dATP and a biotinylated probe on the dTTP supplied for the synthesisof the poly d(A-T), followed by passage of the reaction products over anavidin bead, such as magnetic beads conjugated to avidin; the presenceof the florescent probe on the avidin-containing bead indicates thatpoly d(A-T) has been formed as the fluorescent probe will stick to theavidin bead only if the fluorescenated dATP is incorporated into acovalent linkage with the biotinylated dTTP; and 3) changes fluorescencepolarization indicating an increase in size. Other means of detectingthe presence of poly d(A-T) include the use of intercalatingfluorescence indicators to monitor the increase in duplex DNA formation.

The advantages of the above dual capture assay for detecting antigenicor nucleic acid targets include:

1) No thermocycling of the sample is required. The polymerase domainsand the dATP and dTTP are incubated at a fixed temperature (generallyabout 70° C.). After 30 minutes of incubation up to 75% of the addeddNTPs are incorporated into poly d(A-T). The lack of thermocycling makesthis assay well suited to clinical laboratory settings; there is no needto purchase a thermocycling apparatus and there is no need to maintainvery precise temperature control.

2) The reaction conditions are simple. The incubation of the boundenzymatic domains is done in a buffer containing 0.5 mM MgCl₂ (higherconcentrations may be used), 2-10 mM Tris-Cl, pH 8.5, approximately 50μM dATP and dTTP. The reaction volume is 10-20 μl and reaction productsare detectable within 10-20 minutes.

3) No reaction is detected unless both the synthetic and nucleaseactivities are present. Thus, a positive result indicates that bothprobes (antibody or oligonucleotide) have recognized their targetsthereby increasing the specificity of recognition by having twodifferent probes bind to the target.

The ability to separate the two enzymatic activities of the DNAP allowsfor exponential increases in poly d(A-T) production. If a DNAP is usedwhich lacks 5′ nuclease activity, such as the Klenow fragment ofDNAPEc1, only a linear or arithmetic increase in poly d(A-T) productionis possible (Setlow et al., J. Biol. Chem. 247:224 (1972)). The abilityto provide an enzyme having 5′ nuclease activity but lacking syntheticactivity is made possible by the disclosure of this invention.

V. CLEAVASE Fragment Length Polymorphism for the Detection of SecondaryStructure

Nucleic acids assume secondary structures which depend on base-pairingfor stability. When single strands of nucleic acids (single-strandedDNA, denatured DNA or RNA) with different sequences, even closelyrelated ones, are allowed to fold on themselves, they assumecharacteristic secondary structures. These differences in structuresaccount for the ability of single strand conformation polymorphism(SSCP) analysis to distinguish between DNA fragments having closelyrelated sequences.

The 5′ nuclease domains of certain DNA polymerases are specificendonucleases that recognize and cleave nucleic acids at specificstructures rather than in a sequence-specific manner (as do restrictionendonucleases). The isolated nuclease domain of DNAPTaq described herein(termed the enzyme CLEAVASE) recognizes the end of a duplex that hasnon-base paired strands at the ends. The strand with the 5′ end iscleaved at the junction between the single strand and the duplex.

FIG. 29 depicts a wild-type substrate and a mutant substrate wherein themutant substrate differs from the wild-type by a single base change (Ato G as indicated). According to the method of the present invention,substrate structures form when nucleic acids are denatured and allowedto fold on themselves (See FIG. 29, steps 1 and 2). The step ofdenaturation may be achieved by treating the nucleic acid with heat, low(<3) or high pH (>10), the use of low salt concentrations, the absenceof cations, chemicals (e.g., urea, formamide) or proteins (e.g.,helicases). Folding or renaturation of the nucleic acid is achieved bylowering of the temperature, addition of salt, neutralization of the pH,withdrawal of the chemicals or proteins.

The manner in which the substrate folds is dependent upon the sequenceof the substrate. The 5′ nucleases of the invention cleave thestructures (See FIG. 29, step 3). The end points of the resultingfragments reflect the locations of the cleavage sites. The cleavageitself is dependent upon the formation of a particular structure, notupon a particular sequence at the cleavage site.

When the 5′ nucleases of the invention cleave a nucleic acid substrate,a collection of cleavage products or fragments is generated. Thesefragments constitute a characteristic fingerprint of the nucleic acidwhich can be detected (e.g., by electrophoresis on a gel (see step 4)).Changes in the sequence of a nucleic acid (e.g., single point mutationbetween a wild-type and mutant gene) alter the pattern of cleavagestructures formed. When the 5′ nucleases of the invention cleave thestructures formed by a wild-type and an altered or mutant form of thesubstrate, the distribution of the cleavage fragments generated willdiffer between the two substrates reflecting the difference in thesequence of the two substrates (See FIG. 39, step 5).

The CLEAVASE enzyme generates a unique pattern of cleavage products fora substrate nucleic acid. Digestion with the CLEAVASE enzyme can be usedto detect single base changes in DNA molecules of great length (e.g.,1.6 kb in length) to produce a characteristic pattern of cleavageproducts. The method of the invention is termed “CLEAVASE FragmentLength Polymorphism” (CFLP method). However, it is noted that theinvention is not limited to the use of the enzyme CLEAVASE; suitableenzymatic cleavage activity may be provided from a variety of sourcesincluding the CLEAVASE enzyme, Taq DNA polymerase, E. coli DNApolymerase I and eukaryotic structure-specific endonucleases (e.g., theyeast RAD2 protein and RAD1/RAD10 complex (Harrington, J. J. and Liener(1994) Genes and Develop. 8:1344), murine FEN-1 endonucleases(Harrington and Liener, supra) and calf thymus 5′ to 3′ exonuclease(Murante, R. S., et al. (1994) J. Biol. Chem. 269:1191). Indeed actualexperimental data is provided herein which demonstrates that numerousenzymes may be used to generate a unique pattern of cleavage productsfor a substrate nucleic acid. Enzymes which are shown herein to besuitable for use in the CFLP method include the CLEAVASE BN enzyme, TaqDNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, E. coli Exo III,and the yeast Rad1/Rad10 complex.

The invention demonstrates that numerous enzymes may be suitable for usein the CFLP method including enzymes which have been characterized inthe literature a being 3′ exonucleases. In order to test whether anenzyme is suitable for use as a cleavage means in the CFLP method (i.e.,capable of generating a unique pattern of cleavage products for asubstrate nucleic acid), the following steps are taken. Carefulconsideration of the steps described below allows the evaluation of anyenzyme (“enzyme X”) for use in the CFLP method.

An initial CFLP reaction is prepared using a previously characterizedsubstrate nucleic acid (for example the 157 nucleotide fragment of exon4 of the human tyrosinase gene (SEQ ID NO:47)). The substrate nucleicacid (approximately 100 fmoles; the nucleic acid template may contain a5′ end or other label to permit easy detection of the cleavage products)is placed into a thin wall microcentrifuge tube in a solution whichcomprises reaction conditions reported to be optimal for thecharacterized activity of the enzyme (i.e., enzyme X). For example, ifthe enzyme X is a DNA polymerase, the initial reaction conditions wouldutilize a buffer which has been reported to be optimal for thepolymerization activity of the polymerase. If enzyme X is not apolymerase, or if no specific components are reported to be needed foractivity, the initial reaction may be assembled by placing the substratenucleic acid in a solution comprising 1×CFLP buffer (10 mM MOPS, 0.05%TWEEN-20, 0.05% NONIDET P-40), pH 7.2 to 8.2, 1 mM MnCl₂.

The substrate nucleic acid is denatured by heating the sample tube to95° C. for 5 seconds and then the reaction is cooled to a temperaturesuitable for the enzyme being tested (e.g., if a thermostable polymeraseis being tested the cleavage reaction may proceed at elevatedtemperatures such as 72° C.; if a mesophilic enzyme is being tested thetube is cooled to 37° C. for the cleavage reaction). Followingdenaturation and cooling to the target temperature, the cleavagereaction is initiated by the addition of a solution comprising 1 to 200units of the enzyme to be tested (i.e., enzyme X; the enzyme may bediluted into 1×CFLP buffer, pH 8.2 if desired).

Following the addition of the enzyme X solution, the cleavage reactionis allowed to proceed at the target temperature for 2 to 5 minutes. Thecleavage reaction is then terminated (this may be accomplished by theaddition of a stop solution (95% formamide, 10 mM EDTA, 0.05%bromophenol blue, 0.05% xylene cyanol)) and the cleavage products areresolved and detected using any suitable method (e.g., electrophoresison a denaturing polyacrylamide gel followed by transfer to a solidsupport and nonisotopic detection). The cleavage pattern generated isexamined by the criteria described below for the CFLP reactionoptimization test.

An enzyme is suitable for use in the CFLP method if it is capable ofgenerating a unique (i.e., characteristic) pattern of cleavage productsfrom a substrate nucleic acid; this cleavage must be shown to bedependent upon the presence of the enzyme. Additionally, an enzyme mustbe able to reproducibly generate the same cleavage pattern when a givensubstrate is cleaved under the same reaction conditions. To test forreproducibility, the enzyme to be evaluated is used in at least twoseparate cleavage reactions run on different occasions using the samereaction conditions. If the same cleavage pattern is obtained on bothoccasions, the enzyme is capable of reproducibly generating a cleavagepattern and is therefore suitable for use in the CFLP method.

When enzymes derived from mesophilic organisms are to be tested in theCFLP reaction they may be initially tested at 37° C. However it may bedesirable to use theses enzymes at higher temperatures in the cleavagereaction. The ability to cleave nucleic acid substrates over a range oftemperatures is desirable when the cleavage reaction is being used todetect sequence variation (i.e., mutation) between different substrates.Strong secondary structures that may dominate the cleavage pattern areless likely to be destabilized by single-base changes and may thereforeinterfere with mutation detection. Elevated temperatures can then beused to bring these persistent structures to the brink of instability,so that the effects of small changes in sequence are maximized andrevealed as alterations in the cleavage pattern. Mesophilic enzymes maybe used at temperatures greater than 37° C. under certain conditionsknown to the art. These conditions include the use of high (i.e.,10-30%) concentrations of glycerol in the reaction conditions.Furthermore, it is noted that while an enzyme may be isolated from amesophilic organism this fact alone does not mean that the enzyme maynot demonstrate thermostability; therefore when testing the suitabilityof a mesophilic enzyme in the CFLP reaction, the reaction should be runat 37° C. and at higher temperatures. Alternatively, mild denaturantscan be used to destablize the nucleic acid substrate at a lowertemperature (e.g., 1-10% formamide, 1-10% DMSO and 1-10% glycerol havebeen used in enzymatic reactions to mimic thermal destablization).

Nucleic acid substrates that may be analyzed using a cleavage means,such as a 5′ nuclease, include many types of both RNA and DNA. Suchnucleic acid substrates may all be obtained using standard molecularbiological techniques. For example, substrates may be isolated from atissue sample, tissue culture cells, bacteria or viruses, may betranscribed in vitro from a DNA template, or may be chemicallysynthesized. Furthermore, substrates may be isolated from an organism,either as genomic material or as a plasmid or similar extrachromosomalDNA, or it may be a fragment of such material generated by treatmentwith a restriction endonuclease or other cleavage agents or it may besynthetic.

Substrates may also be produced by amplification using the PCR. When thesubstrate is to be a single-stranded substrate molecule, the substratemay be produced using the PCR with preferential amplification of onestrand (asymmetric PCR). Single-stranded substrates may also beconveniently generated in other ways. For example, a double-strandedmolecule containing a biotin label at the end of one of the two strandsmay be bound to a solid support (e.g., a magnetic bead) linked to astreptavidin moiety. The biotin-labeled strand is selectively capturedby binding to the streptavidin-bead complex. It is noted that thesubsequent cleavage reaction may be performed using substrate attachedto the solid support, as the enzyme CLEAVASE can cleave the substratewhile it is bound to the bead. A single-stranded substrate may also beproduced from a double-stranded molecule by digestion of one strand withexonuclease.

The nucleic acids of interest may contain a label to aid in theirdetection following the cleavage reaction. The label may be aradioisotope (e.g., a ³²P or ³⁵S-labeled nucleotide) placed at eitherthe 5′ or 3′ end of the nucleic acid or alternatively the label may bedistributed throughout the nucleic acid (i.e., an internally labeledsubstrate). The label may be a nonisotopic detectable moiety, such as afluorophore which can be detected directly, or a reactive group whichpermits specific recognition by a secondary agent. For example,biotinylated nucleic acids may be detected by probing with astreptavidin molecule which is coupled to an indicator (e.g., alkalinephosphatase or a fluorophore), or a hapten such as digoxigenin may bedetected using a specific antibody coupled to a similar indicator.Alternatively, unlabeled nucleic acid may be cleaved and visualized bystaining (e.g., ethidium bromide staining) or by hybridization using alabeled probe. In a preferred embodiment, the substrate nucleic acid islabeled at the 5′ end with a biotin molecule and is detected usingavidin or streptavidin coupled to alkaline phosphatase. In anotherpreferred embodiment the substrate nucleic acid is labeled at the 5′ endwith a fluorescein molecule and is detected using an anti-fluoresceinantibody-alkaline phosphatase conjugate.

The cleavage patterns are essentially partial digests of the substratein the reaction. When the substrate is labelled at one end (e.g., withbiotin), all detectable fragments share a common end. The extension ofthe time of incubation of the enzyme CLEAVASE reaction does notsignificantly increase the proportion of short fragments, indicatingthat each potential cleavage site assumes either an active or inactiveconformation and that there is little inter-conversion between thestates of any potential site, once they have formed. Nevertheless, manyof the structures recognized as active cleavage sites are likely to beonly a few base-pairs long and would appear to be unstable at theelevated temperatures used in the CLEAVASE enzyme reaction. Theformation or disruption of these structures in response to smallsequence changes results in changes in the patterns of cleavage.

The products of the cleavage reaction are a collection of fragmentsgenerated by structure specific cleavage of the input nucleic acid.Nucleic acids which differ in size may be analyzed and resolved by anumber of methods including electrophoresis, chromatography,fluorescence polarization, mass spectrometry and chip hybridization. Theinvention is illustrated using electrophoretic separation. However, itis noted that the resolution of the cleavage products is not limited toelectrophoresis. Electrophoresis is chosen to illustrate the method ofthe invention because electrophoresis is widely practiced in the art andis easily accessible to the average practitioner.

If abundant quantities of DNA are available for the analysis, it may beadvantageous to use direct fluorescence to detect the cleavagefragments, raising the possibility of analyzing several samples in thesame tube and on the same gel. This “multiplexing” would permitautomated comparisons of closely related substrates such as wild-typeand mutant forms of a gene.

The CFLP reaction is useful to rapidly screen for differences betweensimilar nucleic acid molecules. To optimize the CFLP reaction for anydesired nucleic acid system (e.g., a wild-type nucleic acid and one ormore mutant forms of the wild-type nucleic acid), it is most convenientto use a single substrate from the test system (for example, thewild-type substrate) to determine the best CFLP reaction conditions. Asingle suitable condition is chosen for doing the comparison CFLPreactions on the other molecules of interest. For example, a cleavagereaction may be optimized for a wild-type sequence and mutant sequencesmay subsequently be cleaved under the same conditions for comparisonwith the wild-type pattern. The objective of the CFLP reactionoptimization test is the identification of a set of conditions whichallow the test molecule to form an assortment (i.e., a population) ofintra-strand structures that are sufficiently stable such that treatmentwith a structure-specific cleavage agent such as the CLEAVASE enzyme orDNAPTaq will yield a signature array of cleavage products, yet aresufficiently unstable that minor or single-base changes within the testmolecule are likely to result in a noticeable change in the array ofcleavage products.

The following discussion illustrates the optimization of the CFLP methodfor use with a single-stranded substrate.

A panel of reaction conditions with varying salt concentration andtemperature is first performed to identify an optimal set of conditionsfor the single-stranded CFLP reaction. “Optimal CFLP” reaction isdefined for this test case as the set of conditions that yields the mostwidely spaced set of bands after electrophoretic separation, with themost even signal intensity between the bands.

Two elements of the cleavage reaction that significantly affect thestability of the nucleic acid structures are the temperature at whichthe cleavage reaction is performed and the concentration of salt in thereaction solution. Likewise, other factors affecting nucleic acidstructures, such as, formamide, urea or extremes in pH may be used. Theinitial test typically will comprise reactions performed at fourtemperatures (60° C., 65° C., 70° C. and 75° C.) in three different saltconcentrations (0 mM, 25 mM and 50 mM) for a total of twelve individualreactions. It is not intended that the present invention be limited bythe salt utilized. The salt utilized may be chosen from potassiumchloride, sodium chloride, etc. with potassium chloride being apreferred salt.

For each salt concentration to be tested, 30 μl of a master mixcontaining a DNA substrate, buffer and salt is prepared. When thesubstrate is DNA, suitable buffers include3-(N-Morpholino)propanesulfonic acid (MOPS), pH 6.5 to 9.0, with pH 7.5to 8.4 being particularly preferred and other “Good” biological bufferssuch as tris(Hydroxymethyl)aminomethane (Tris) orN,N-bis(2-Hydroxyethyl)glycine (Bicine), pH 6.5 to 9.0, with pH 7.5 to8.4 being particularly preferred. When the nucleic acid substrate isRNA, the pH of the buffer is reduced to the range of 6.0 to 8.5, with pH6.0 to 7.0 being particularly preferred. When manganese is to used asthe divalent cation in the reaction, the use of Tris buffers is notpreferred. Manganese tends to precipitate as manganous oxide in Tris ifthe divalent cation is exposed to the buffer for prolonged periods (suchas in incubations of greater than 5 minutes or in the storage of a stockbuffer). When manganese is to be used as the divalent cation, apreferred buffer is the MOPS buffer.

For reactions containing no salt (the “0 mM KCl” mix), the mix includesenough detectable DNA for 5 digests (e.g., approximately 500 fmoles of5′ biotinylated DNA or approximately 100 fmoles of ³²P-5′ end labeledDNA) in 30 μl of 1×CFLP buffer (10 mM MOPS, pH 8.2) with 1.7 mM MnCl₂ orMgCl₂ (the final concentration of the divalent cation will be 1 mM).Other concentrations of the divalent cation may be used if appropriatefor the cleavage agent chosen (e.g., E. coli DNA polymerase I iscommonly used in a buffer containing 5 mM MgCl₂). The “25 mM KCl” mixincludes 41.5 mM KCl in addition to the above components; the “50 mMKCl” mix includes 83.3 mM KCl in addition to the above components.

The mixes are distributed into labeled reaction tubes (0.2 ml, 0.5 ml or1.5 ml “Eppendorf” style microcentrifuge tubes) in 6 μl aliquots,overlaid with light mineral oil or a similar barrier, and stored on iceuntil use. Sixty microliters of an enzyme dilution cocktail isassembled, comprising a 5′ nuclease at a suitable concentration in1×CFLP buffer without MnCl₂. Preferred 5′ nucleases and concentrationsare 750 ng of the enzyme CLEAVASE BN or 15 units of Taq DNA polymerase(or another eubacterial Pol A-type DNA polymerase). Suitable amounts ofa similar structure-specific cleavage agent in 1×CFLP buffer withoutMnCl₂ may also be utilized.

If a strong (i.e., stable) secondary structure is formed by thesubstrates, a single nucleotide change is unlikely to significantlyalter that structure, or the cleavage pattern it produces. Elevatedtemperatures can be used to bring structures to the brink ofinstability, so that the effects of small changes in sequence aremaximized, and revealed as alterations in the cleavage pattern withinthe target substrate, thus allowing the cleavage reaction to occur atthat point. Consequently, it is often desirable to run the reaction atan elevated temperature (i.e., above 55° C.).

Preferably, reactions are performed at 60° C., 65° C., 70° C. and 75° C.For each temperature to be tested, a trio of tubes at each of the threeKCl concentrations are brought to 95° C. for 5 seconds, then cooled tothe selected temperature. The reactions are then started immediately bythe addition of 4 μl of the enzyme cocktail. A duplicate trio of tubesmay be included (these tubes receiving 4 μl of 1×CFLP buffer withoutenzyme or MnCl₂), to assess the nucleic acid stability in these reactionconditions. All reactions proceed for 5 minutes, and are stopped by theaddition of 8 μl of 95% formamide with 20 mM EDTA and 0.05% xylenecyanol and 0.05% bromophenol blue. Reactions may be assembled and storedon ice if necessary. Completed reactions are stored on ice until allreactions in the series have been performed.

Samples are heated to 72° C. for 2 minutes and 5 μl of each reaction isresolved by electrophoresis through a suitable gel, such as 6 to 10%polyacrylamide (19:1 cross-link), with 7M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA for nucleic acids up to approximately1.5 kb, or native or denaturing agarose gels for larger molecules. Thenucleic acids may be visualized as described above, by staining,autoradiography (for radioisotopes) or by transfer to a nylon or othermembrane support with subsequent hybridization and/or nonisotopicdetection. The patterns generated are examined by the criteria describedabove and a reaction condition is chosen for the performance of thevariant comparison CFLP patterns.

A “no enzyme” control allows the assessment of the stability of thenucleic acid substrate under particular reaction conditions. In thisinstance, the substrate is placed in a tube containing all reactioncomponents except the enzyme and treated the same as theenzyme-containing reactions. Other control reactions may be run. Awild-type substrate may be cleaved each time a new mutant substrate istested. Alternatively, a previously characterized mutant may be run inparallel with a substrate suspected of containing a different mutation.Previously characterized substrates allow for the comparison of thecleavage pattern produced by the new test substrate with a knowncleavage pattern. In this manner, alterations in the new test substratemay be identified.

When the CFLP pattern generated by cleavage of a single-strandedsubstrate contains an overly strong (i.e., intense) band, this indicatesthe presence of a very stable structure. The preferred method forredistributing the signal is to alter the reaction conditions toincrease structure stability (e.g., lower the temperature of thecleavage reaction, raise the monovalent salt concentration); this allowsother less stable structures to compete more effectively for cleavage.

When the single-stranded substrate is labelled at one end (e.g., withbiotin or 32P) all detectable fragments share a common end. For shortDNA substrates (less than 250 nucleotides) the concentration of theenzyme (e.g., CLEAVASE BN) and the length of the incubation have minimalinfluence on the distribution of signal intensity, indicating that thecleavage patterns are not partial digests of a single structure assumedby the nucleic acid substrate, but rather are relatively completedigests of a collection of stable structures formed by the substrate.With longer DNA substrates (greater than 250 nucleotides) there is agreater chance of having multiple cleavage sites on each structure,giving apparent overdigestion as indicated by the absence of anyresidual full-length materials. For these DNA substrates, the enzymeconcentration may be lowered in the cleavage reaction (for example, if50 ng of the CLEAVASE BN enzyme were used initially and overdigestionwas apparent, the concentration of enzyme may be reduced to 25, 10 or 1ng per reaction).

When the CFLP reaction is to optimized for the cleavage adouble-stranded substrate the following steps are taken. The cleavage ofdouble-stranded DNA substrates up to 2,000 base pairs may be optimizedin this manner.

The double-stranded substrate is prepared such that it contains a singleend-label using any of the methods known to the art. The molar amount ofDNA used in the optimization reactions is the same as that use for theoptimization of reactions utilizing single-stranded substrates. The mostnotable differences between the optimization of the CFLP reaction forsingle-versus double-stranded substrates is that the double-strandedsubstrate is denatured in distilled water without buffer, theconcentration of MnCl₂ in the reaction is reduced to 0.2 mM, the KCl (orother monovalent salt) is omitted, and the enzyme concentration isreduced to 10 to 25 ng per reaction. In contrast to the optimization ofthe single-stranded CFLP reaction (described above) where the variationof the monovalent salt (e.g., KCl) concentration is a criticalcontrolling factor, in the optimization of the double-stranded CFLPreaction the range of temperature is the more critical controllingfactor for optimization of the reaction. When optimizing thedouble-stranded CFLP reaction a reaction tube containing the substrateand other components described below is set up to allow performance ofthe reaction at each of the following temperatures: 40° C., 45° C., 50°C., 55° C., 60° C., 65° C., 70° C., and 75° C.

For each temperature to be tested, a mixture comprising the single endlabelled double-stranded DNA substrate and distilled water in a volumeof 15 μl is prepared and placed into a thin walled microcentrifuge tube.This mixture may be overlaid with light mineral oil or liquid wax (thisoverlay is not generally required but may provide more consistentresults with some double-stranded DNA substrates).

A 2 mM solution of MnCl₂ is prepared. For each CFLP reaction, 5 μl of adiluted enzyme solution is prepared comprising 2 μl of 10×CFLP buffer(100 mM MOPS, pH 7.2 to 8.2, 0.5% TWEEN-20, 0.5% NONIDET P-40), 2 μl of2 mM MnCl₂ and 25 ng of CLEAVASE BN enzyme and distilled water to yielda final volume of 5 μl.

The DNA mixture is heated to 95° C. for 10 to 30 seconds and thenindividual tubes are cooled to the reaction temperatures to be tested(e.g., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., and 75°C.). The cleavage reaction is started by adding 5 μl of the diluteenzyme solution to each tube at the target reaction temperature. Thereaction is incubated at the target temperature for 5 minutes and thereaction is terminated (e.g., by the addition of 16 μl of stop solutioncomprising 95% formamide with 10 mM EDTA and 0.05% xylene cyanol and0.05% bromophenol blue).

Samples are heated to 72° C. for 1 to 2 minutes and 3 to 7 μl of eachreaction is resolved by electrophoresis through a suitable gel, such as6 to 10% polyacrylamide (19:1 cross-link), with 7M urea, in a buffer of45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA for nucleic acids up toapproximately 1.5 kb, or native or denaturing agarose gels for largermolecules. The nucleic acids may be visualized as described above, bystaining, autoradiography (for radioisotopes) or by transfer to a nylonor other membrane support with subsequent hybridization and/ornonisotopic detection. The patterns generated are examined by thecriteria described above and a reaction condition is chosen for theperformance of the double-stranded CFLP reaction.

A “no enzyme” control allows the assessment of the stability of thenucleic acid substrate under particular reaction conditions. In thisinstance, the substrate is placed in a tube containing all reactioncomponents except the enzyme and treated the same as theenzyme-containing reactions. Other control reactions may be run. Awild-type substrate may be cleaved each time a new mutant substrate istested. Alternatively, a previously characterized mutant may be run inparallel with a substrate suspected of containing a different mutation.Previously characterized substrates allow for the comparison of thecleavage pattern produced by the new test substrate with a knowncleavage pattern. In this manner, alterations in the new test substratemay be identified.

When performing double-stranded CFLP reactions the MnCl₂ concentrationpreferably will not exceed 0.25 mM. If the end label on thedouble-stranded DNA substrate disappears (i.e., loses its 5′ end labelas judged by a loss of signal upon detection of the cleavage products),the concentration of MnCl₂ may be reduced to 0.1 mM. Any EDTA present inthe DNA storage buffer will reduce the amount of free Mn²⁺ in thereaction, so double-stranded DNA should be dissolved in water orTris-HCl with a EDTA concentration of 0.1 mM or less.

Cleavage products produced by cleavage of either single-ordouble-stranded substrates which contain a biotin label may be detectedusing the following nonisotopic detection method. After electrophoresisof the reaction products, the gel plates are separated allowing the gelto remain flat on one plate. A positively charged nylon membrane(preferred membranes include NYTRAN Plus, 0.2 or 0.45 mm-pore size,Schleicher and Schuell, Keene, N.H.), cut to size and pre-wetted in0.5×TBE (45 mM tris-Borate, pH 8.3, 1.4 mM EDTA), is laid on top of theexposed gel. All air bubbles trapped between the gel and the membraneare removed (e.g., by rolling a 10 ml pipet firmly across the membrane).Two pieces of 3MM filter paper (Whatman) are then placed on top of themembrane, the other glass plate is replaced, and the sandwich is clampedwith binder clips or pressed with books or weights. The transfer isallowed to proceed 2 hours to overnight (the signal increases withlonger transfer).

After transfer, the membrane is carefully peeled from the gel andallowed to air dry. Distilled water from a squeeze bottle can be used toloosen any gel that sticks to the membrane. After complete drying, themembrane is agitated for 30 minutes in 1.2× SEQUENASE IMAGES BlockingBuffer (United States Biochemical, Cleveland, Ohio; avoid anyprecipitates in the blocking buffer by decanting or filtering); 0.3 mlof the buffer is used per cm² of membrane (e.g., 30 mls for a 10 cm×10cm blot). A streptavidin-alkaline phosphatase conjugate (SAAP, UnitedStated Biochemical) is added at a 1:4000 dilution directly to theblocking solution (avoid spotting directly on membrane), and agitatedfor 15 minutes. The membrane is rinsed briefly with dH₂O and then washed3 times (5 minutes of shaking per/wash) in 1×SAAP buffer (100 mMTris-HCl, pH 10; 50 mM NaCl) with 0.1% sodium dodecyl sulfate (SDS),using 0.5 ml buffer/cm² of membrane, with brief water rinses betweeneach wash. The membrane is then washed twice in 1×SAAP buffer (no SDS)with 1 mM MgCl₂, drained thoroughly, and placed in a plasticheat-sealable bag. Using a sterile pipet tip, 0.05 ml/cm² of CDPSTARreagent (Tropix, Bedford, Mass.) is added to the bag and distributedover the entire membrane for 5 minutes. The bag is drained of all excessliquid and air bubbles, sealed, and the membrane is exposed to X-rayfilm (e.g., Kodak XRP) for 30 minutes. Exposure times are adjusted asnecessary for resolution and clarity.

To date, every nucleic acid substrate tested in the CFLP system hasproduced a reproducible pattern of fragments. The sensitivity andspecificity of the cleavage reaction make this method of analysis verysuitable for the rapid screening of mutations in cancer diagnostics,tissue typing, genetic identity, bacterial and viral typing,polymorphism analysis, structure analysis, mutant screening in geneticcrosses, etc. It could also be applied to enhanced RNA analysis, highlevel multiplexing and extension to longer fragments. One distinctbenefit of using the CLEAVASE enzyme reaction to characterize nucleicacids is that the pattern of cleavage products constitutes acharacteristic fingerprint, so a potential mutant can be compared topreviously characterized mutants without sequencing. Also, the place inthe fragment pattern where a change is observed gives a good indicationof the position of the mutation. But it is noted that the mutation neednot be at the precise site of cleavage, but only in an area that affectsthe stability of the structure.

VI. Detection of Mutations in the p53 Tumor Suppressor Gene Using theCFLP Method

Tumor supressor genes control cellular proliferation and a variety ofother processes important for tissue homeostasis. One of the mostextensively studied of these, the p53 gene, encodes a regulator of thecell cycle machinery that can suppress the growth of cancer cells aswell as inhibit cell transformation (Levine, Annu Rev. Biochem. 62:623(1993)). Tumor supressor mutations that alter or obliterate normal p53function are common.

Mutations in the p53 tumor supressor gene are found in about half of allcases of human cancer making alterations in the p53 gene the most commoncancer-related genetic change known at the gene level. In the wild-typeor non-mutated form, the p53 gene encodes a 53-kD nuclearphosphoprotein, comprising 393 amino acids, which is involved in thecontrol of cellular proliferation. Mutations in the p53 gene aregenerally (greater than 90%) missense mutations which cause a change inthe identity of an amino acid rather than nonsense mutations which causeinactivation of the protein. It has been postulated that the highfrequency of p53 mutation seen in human tumors is due to the fact thatthe missense mutations cause both a loss of tumor supressor function anda gain of oncogenic function (Lane, D. P. and Benchimol, S., Genes Dev.4:1 (1990)).

The gene encoding the p53 protein is large, spanning 20,000 base pairs,and is divided into 11 exons (see FIG. 76). The ability to scan thelarge p53 gene for the presence of mutations has important clinicalapplications. In several major human cancers the presence of a tumor p53mutation is associated with a poor prognosis. p53 mutation has beenshown to be an independent marker of reduced survival in lymphnode-negative breast cancers, a finding that may assist clinicians inreaching decisions regarding more aggressive therapeutic treatment.Also, Lowe and co-workers have demonstrated that the vulnerability oftumor cells to radiation or chemotherapy is greatly reduced by mutationswhich abolish p53-dependent apoptosis (Lowe et al., Cell 74:957 (1995)).

Regions of the p53 gene from approximately 10,000 tumors have beensequenced in the last 4 to 5 years, resulting in characterization ofover 3,700 mutations of which approximately 1,200 represent independentp53 mutations (i.e., point mutations, insertion or deletions). Adatabase has been compiled and deposited with the European MolecularBiology Laboratory (EMBL) Data Library and is available in electronicform (Hollstein, M. et al. (1994) Nucleic Acids Res. 22:3551 andCariello, N. F. et al. (1994) Nucleic Acids Res. 22:3549). In addition,an IBM PC compatible software package to analyze the information in thedatabase has been developed. (Cariello et al., Nucel. Acids Res. 22:3551(1994)). The point mutations in the database were identified by DNAsequencing of PCR-amplified products. In most cases, preliminaryscreening for mutations by SSCP or DGGE was performed.

Analysis of the p53 mutations shows that the p53 gene contains 5 hotspot regions (HSR) most frequently mutated in human tumors that show atight correlation between domains of the protein that are evolutionaryhighly conserved (ECDs) and seem to be specifically involved in thetransformation process (see FIG. 76; the height of the bar represent therelative percentage of total mutations associated with the five HSRs).The five HSRs are confined to exons 5 to 8 and account for over 85% ofthe mutations detected. However, because these studies generallyconfined their analysis to PCR amplifications and sequencing of regionslocated between exons 5 to 8, it should be kept in mind that mutationsoutside this region are underrepresented. As 10% to 15% of the mutationslie outside this region, a clinically effective p53 gene DNA diagnosticshould be able to cost-effectively scan for life-threatening mutationsscattered across the entire gene (33).

The following table lists a number of the known p53 mutations.

TABLE 2 HUMAN p53 GENE MUTATIONS CODON NO. WILD-TYPE MUTANT EVENT TUMORTYPE 36 CCG CCA GC→AT Lung 49 GAT CAT GC→CG CML 53 TGG TGT GC→TA CML 60CCA TCA GC→AT CML 68 GAG TAG GC→TA SCLC 110 CGT TGT GC→AT Hepatoca 113TTC TGT Double M NSCLC 128 CCT CCG T→G Breast 128 TCT C→T Breast 129 GCCGAC GC→TA Neurofibrosa 130 CTC CTG GC→CG MDS 132 AAG AAC GC→CGColorectal ca 132 CAG AT→CG Breast ca 132 AAT GC→TA Lung (NSCLC) ca 132CAG AT→CG Pancreatic ca 132 AGG AT→GC CML 133 ATG TTG AT→TA Colorectalca 133 AAG AT→TA Burkitt lymphoma 134 TTT TTA AT→TA Lung (SCLC) ca 135TGC TAC GC→AT Colorectal ca 135 TCC GC→CG AML 135 TAC GC→AT Lung (NSCLC)ca 135 TGG GC→CG MDS 136 CAA GAG Double M Breast ca 138 GCC GTC GC→ATRhabdomyosa 138 GGC GC→CG Lung (SCLC) ca 140 ACC TAC AT→TA CML 141 TGCTAC GC→AT Colorectal ca 141 TAC GC→AT Bladder ca 143 GTG GCG AT→GCColorectal ca 143 TTG GC→TA Lung (NSCLC) ca 144 CAG TAG GC→AT Esophagealca 144 CCG AT→CG Burkitt lymphoma 151 CCC CAT Double M Leiomyosa 151 CACGC→TA Lung (SCLC) ca 151 TCC GC→AT Glioblastoma 151 TCC GC→AT Lung(NSCLC) ca 152 CCG CTG GC→AT Leiomyosa 152 TCG GC→AT Breast ca 154 GGCGTC GC→TA Esophageal ca 154 GTC GC→TA Lung (NSCLC) ca 154 GTC GC→TA Lung(NSCLC) ca 154 GTC GC→TA Lung (NSCLC) ca 156 CGC CCC GC→CG Rhabdomyosa156 CCC GC→CG Osteosa 156 CGT GC→AT Lung (NSCLC) ca 156 CCC GC→CG Lung(NSCLC) ca 157 GTC TTC GC→TA Hepatoca 157 TTC GC→TA Lung (SCLC) ca 157TTC GC→TA Lung (NSCLC) ca 157 TTC GC→TA Breast ca 157 TTC GC→TA Lung(SCLC) ca 157 TTC GC→TA Bladder ca 158 CGC CGT GC→AT Neurofibrosa 158CAC GC→AT Burkitt lymphoma 159 GCC GTC GC→AT Lung (NSCLC) ca 159 CCCGC→CG Lung (NSCLC) ca 163 TAC TGC AT→GC Breast ca 163 CAC AT→GC Burkittlymphoma 164 AAG CAG AT→CG Breast ca 171 GAG TAG GC→TA Lung (SCLC) ca172 GTT TTT GC→TA Burkitt lymphoma 173 GTG TTG GC→TA Lung (NSCLC) ca 173TTG GC→TA Lung (NSCLC) ca 173 GGG AT→CG Burkitt lymphoma 173 GTA GC→ATGastric ca 175 CGC CAC GC→AT Colorectal ad 175 CAC GC→AT Colorectal ad175 CAC GC→AT Colorectal ad 175 CAC GC→AT Colorectal ca 175 CAC GC→ATColorectal ca 175 CAC GC→AT T-ALL 175 CAC GC→AT Brain tumor 175 CACGC→AT Colorectal ca 175 CAC GC→AT Colorectal ca 175 CAC GC→AT Leiomyosa175 CAC GC→AT Esophageal ca 175 CAC GC→AT Glioblastoma 175 CAC GC→ATColorectal ca 175 CAC GC→AT T-ALL 175 CAC GC→AT Breast ca 175 CTC GC→TABreast ca 175 AGC GC→TA Hepatoca 175 CAC GC→AT B-ALL 175 CAC GC→AT B-ALL175 CAC GC→AT Burkitt lymphoma 175 CAC GC→AT Burkitt lymphoma 175 CACGC→AT Burkitt lymphoma 175 CAC GC→AT Burkitt lymphoma 175 CAC GC→ATGastric ca 176 TGC TTC GC→TA Lung (NSCLC) ca 176 TTC GC→TA Esophageal ca176 TTC GC→TA Lung (NSCLC) ca 176 TAC GC→AT Burkitt lymphoma 177 CCC CGCGC→CG PTLC 179 CAT TAT GC→AT Neurofibrosa 179 CAG AT→CG Lung (SCLC) ca179 CTT AT→TA Esophageal ca 179 GAT GC→CG Breast ca 179 CTT AT→TACholangiosa 179 CTT AT→TA Cholangiosa 181 CGC CAC GC→AT Li-Fraumeni sdm187 GGT TGT GC→TA Breast ca 192 CAG TAG GC→AT Esophageal ca 193 CAT CGTAT→GC Lung (SCLC) ca 193 TAT GC→AT Esophageal ca 193 CGT AT→GC AML 194CTT TTT GC→AT Breast ca 194 CGT AT→CG Lung (SCLC) ca 194 CGT AT→CGEsophageal ca 194 CGT AT→CG Esophageal ca 194 CGT AT→CG B-CLL 196 CGATGA GC→AT Colorectal ca 196 TGA GC→AT T-ALL 196 TGA GC→AT T-celllymphoma 196 TGA GC→AT Lung (SCLC) ca 196 TGA GC→AT Bladder ca 198 GAATAA GC→TA Lung (SCLC) ca 198 TAA GC→TA Lung (SCLC) ca 202 CGT CTT GC→TACML 204 GAG GGG AT→GC CML 205 TAT TGT AT→GC B-ALL 205 TGT AT→GC B-CLL205 TTT AT→TA Gastric ca 211 ACT GCT AT→GC Colorectal ca 213 CGA TGAGC→AT Colorectal ca 213 CAA GC→AT B-cell lymphoma 213 CAA GC→AT Burkittlymphoma 213 CGG AT→GC Lung (SCLC) ca 213 CGG AT→GC Esophageal ca 213TGA GC→AT Lung (NSCLC) ca 213 CGG AT→GC Lung (NSCLC) ca 213 TGA GC→ATBurkitt lymphoma 213 TGA GC→AT Burkitt lymphoma 215 AGT GGT AT→GCColorectal ca 216 GTG ATG GC→AT Brain tumor 216 GAG AT→TA Burkittlymphoma 216 TTG GC→TA Gastric ca 216 ATG GC→AT Ovarian ca 220 TAT TGTAT→GC Colorectal ca 229 TGT TGA AT→TA Lung (SCLC) ca 232 ATC AGC AT→CGB-CLL 234 TAC CAC AT→GC B-cell lymphoma 234 CAC AT→GC Burkitt lymphoma234 TGC AT→GC Burkitt lymphoma 236 TAC TGC AT→GC Burkitt lymphoma 237ATG AGG AT→CG T-ALL 237 ATA GC→AT Lung (SCLC) ca 237 ATA GC→AT AML 237ATA GC→AT Breast ca 237 ATA GC→AT Burkitt lymphoma 237 ATA GC→ATRichter's sdm 238 TGT TTT GC→TA Larynx ca 238 TAT GC→AT Burkitt lymphoma238 TAT GC→AT CML 239 AAC AGC AT→GC Colorectal ca 239 AGC AT→GCColorectal ca 239 AGC AT→GC Burkitt lymphoma 239 AGC AT→GC CML 239 AGCAT→GC CML 239 AGC AT→GC B-CLL 241 TCC TTC GC→AT Colorectal ca 241 TGCGC→CG Colorectal ca 241 TGC GC→CG Bladder ca 242 TGC TCC GC→CG Lung(SCLC) ca 242 TTC GC→TA Breast ca 242 TCC GC→CG MDS 242 TAC GC→ATEpendymoma 244 GGC TGC GC→TA T-ALL 244 TGC GC→TA Esphageal ca 244 TGCGC→TA Lung (SCLC) ca 244 AGC GC→AT Hepatoca 245 GGC GTC GC→TA Esophagealca 245 TGC GC→TA Li-Fraumeni sdm 245 AGC GC→AT Leyomyosa 245 GAC GC→ATLi-Fraumeni sdm 245 AGC GC→AT Esophageal ca 245 GCC GC→CG Bladder ca 245GAC GC→AT Breast ca 245 GAC GC→AT Li-Fraumeni sdm 245 GGC TGC GC→TALi-Fraumeni sdm 245 GTC GC→TA Cervical ca 246 ATG GTG AT→GC AML 246 ATCGC→CG Lung (NSCLC) ca 246 GTG AT→GC Hepatoca 246 GTG AT→GC Bladder ca247 AAC ATC AT→TA Lung (NSCLC) ca 248 CGG TGG GC→AT Colorectal ad 248TGG GC→AT Colorectal ca 248 CAG GC→AT Colorectal ca 248 CAG GC→ATColorectal ca 248 CAG GC→AT T-ALL 248 CAG GC→AT Esophageal ca 248 TGGGC→AT Li-Fraumeni sdm 248 TGG GC→AT Li-Fraumeni sdm 248 TGG GC→ATColorectal ca 248 TGG GC→AT Colorectal ca 248 TGG GC→AT Rhabdomyosa 248CTG GC→TA Esophageal ca 248 TGG GC→AT Lung (NSCLC) ca 248 CAG GC→AT Lung(SCLC) ca 248 CTG GC→TA Lung (SCLC) ca 248 CAG GC→AT T-ALL 248 TGG GC→ATLung (NSCLC) ca 248 CTG GC→TA Lung (SCLC) ca 248 TGG GC→AT Colorectal ca248 CAG GC→AT Bladder ca 248 CAG GC→AT MDS 248 TGG GC→AT Burkittlymphoma 248 CAG GC→AT Breast ca 248 CAG GC→AT B-CLL 248 CAG GC→ATBurkitt lymphoma 248 TGG GC→AT Burkitt lymphoma 248 CAG GC→AT Burkittlymphoma 248 TGG GC→AT Burkitt lymphoma 248 CAG GC→AT Gastric ca 248 TGGGC→AT Lung (SCLC) ca 248 CAG GC→AT Breast ca 248 CAG GC→AT CML 248 TGGGC→AT Li-Fraumeni sdm 248 CAG GC→AT Li-Fraumeni sdm 248 TGG GC→ATColorectal ca 249 AGG AGT GC→TA Hepatoca 249 AGT GC→TA Hepatoca 249 AGTGC→TA Hepatoca 249 AGC GC→CG Hepatoca 249 AGT GC→TA Hepatoca 249 AGTGC→TA Hepatoca 249 AGT GC→TA Hepatoca 249 AGT GC→TA Hepatoca 249 AGTGC→TA Hepatoca 249 AGT GC→TA Hepatoca 249 AGT GC→TA Hepatoca 249 AGTGC→TA Esophageal ca 249 AGC GC→CG Breast ca 249 AGT GC→TA Lung (NSCLC)ca 249 AGT GC→TA Hepatoca 250 CCC CTC GC→AT Burkitt lymphoma 251 ATC AGCAT→CG Gastric ca 252 CTC CCC AT→GC Li-Fraumeni sdm 252 CTC CCC AT→GCLi-Fraumeni sdm 254 ATC GAC Double M Burkitt lymphoma 254 AAC AT→TABreast ca 256 ACA GCA AT→GC T-ALL 258 GAA AAA GC→AT Li-Fraumeni sdm 258AAA GC→AT Burkitt lymphoma 258 AAA GC→AT Li-Fraumeni sdm 259 GAC GGCAT→GC T-ALL 260 TCC GCC AT→CG T-ALL 266 GGA GTA GC→TA Lung (NSCLC) ca266 GTA GC→TA Lung (NSCLC) ca 266 GTA GC→TA Breast ca 267 CGG CCG GC→CGLung (SCLC) ca 270 TTT TGT AT→CG Esophageal ca 270 TGT AT→CG T-ALL 272GTG ATG GC→AT Brain tumor 272 CTG GC→CG Lung (SCLC) ca 272 ATG GC→ATHepatoca 272 ATG GC→AT AML 273 CGT TGT GC→AT Colorectal ad 273 TGT GC→ATBrain tumor 273 CAT GC→AT Breast ca 273 CAT GC→AT Colorectal ca 273 TGTGC→AT Lung (NSCLC) ca 273 CTT GC→TA Lung (SCLC) ca 273 CAT GC→ATColorectal ca 273 CAT GC→AT Colorectal ca 273 CAT GC→AT Colorectal ca273 CAT GC→AT Lung (NSCLC) ca 273 CCT GC→CG Lung (NSCLC) ca 273 CTTGC→TA Lung (NSCLC) ca 273 CTT GC→TA Lung (NSCLC) ca 273 CAT GC→ATThyroid ca 273 CAT GC→AT Lung (SCLC) ca 273 TGT GC→AT B-cell lymphoma273 TGT GC→AT B-ALL 273 TGT GC→AT Burkitt lymphoma 273 TGT GC→AT Burkittlymphoma 273 CAT GC→AT Li-Fraumeni sdm 273 TGT GC→AT Cervical ca 273 TGTGC→AT AML 273 CAT GC→AT B→CLL 273 CTT GC→TA B-CLL 274 GTT GAT AT→TAErythroleukemia 276 GCC CCC GC→CG B-ALL 276 GAC GC→TA Hepatoca 277 TGTTTT GC→TA Lung (SCLC) ca 278 CCT TCT GC→AT Esophageal ca 278 CTT GC→ATEsophageal ca 278 GCT GC→CG Breast ca 278 TCT GC→AT Lung (SCLC) ca 278CGT GC→CG Ovarian ca 280 AGA AAA GC→AT Esophageal ca 280 AAA GC→ATBreast ca 281 GAC GGC AT→GC Colorectal ca 281 GGC AT→GC Breast ca 281GAC GAG GC→CG Richter's sdm 281 TAC GC→TA B-CLL 282 CGG TGG GC→ATColorectal ad 282 TGG GC→AT Colorectal ca 282 CGG TGG GC→AT Rhabdomyosa282 GGG GC→CG Lung (NSCLC) ca 282 CCG GC→CG Breast ca 282 TGG GC→ATBladder ca 282 TGG GC→AT AML 282 CTG GC→TA Breast ca 282 TGG GC→AT B-ALL282 TGG GC→AT Burkitt lymphoma 282 TGG GC→AT Richter's sdm 282 TGG GC→ATOvarian ca 282 TGG GC→AT Li-Fraumeni sdm 283 CGC TGC GC→AT Colorectal ca283 CCC GC→CG Lung (NSCLC) ca 285 GAG AAG GC→AT Breast ca 286 GAA AAAGC→AT Colorectal ca 286 GGA AT→GC Lung (SCLC) ca 286 GCA AT→CGLi-Fraumeni sdm 287 GAG TAG GC→TA Burkitt lymphoma 293 GGG TGG GC→TAGlioblastoma 298 GAG TAG GC→TA Bladder ca 302 GGG GGT GC→TA Lung (SCLC)ca 305 AAG TAG AT→TA Esophageal ca 305 TAG AT→TA Esophageal ca 307 GCAACA GC→AT Breast ca 309 CCC TCC GC→AT Colorectal ca 334 GGG GTG GC→TALung (SCLC) ca 342 CGA TGA GC→AT Lung (SCLC) ca

DELETIONS/INSERTIONS CODON EVENT TUMOR TYPE 137 del 7 Gastric ca 143 del1 Gastric ca 152 del 13 Colorectal ad 167 del 1 Breast ca 168 del 31Hepatoca 175 del 18 Breast ca 190 del 3 nu1 ALL 201 del 1 Breast ca 206del 1 Burkitt lymphoma 206 del 1 Burkitt lymphoma 214 del 1 B-ALL 236del 27 Bladder ca 239 del 1 Lung (NSCLC) ca 262 del 1 Astrocytoma 262del 24 Gastric ca 262 del 24 Lung (NSCLC) ca 263 del 1 Esophageal ca 264del 1 AML 286 del 8 Hepatoca 293 del 1 Lung (NSCLC) ca 307 del 1Li-Fraumeni sdm 381 del 1 Hepatoca Exon 5 del 15 B-ALL 152 ins 1 B-CLL239 ins 1 Waldenstrom sdm 252 ins 4 Gastric ca 256 ins 1 AML 275 ins 1B-CLL 301 ins 1 MDS 307 ins 1 Glioblastoma Exon 8 ins 25 HCL

SPLICE MUTATIONS INTRON SITE EVENT TUMOR TYPE Intron 3 Accept GC→CG Lung(SCLC) ca Intron 4 Donor GC→TA Lung (SCLC) ca Intron 4 Donor GC→AT T→ALLIntron 5 Donor GC→AT CML Intron 6 Donor AT→CG Lung (SCLC) ca Intron 6Accept AT→TA Lung (SCLC) ca Intron 6 Accept AT→TA Lung (NSCLC) ca Intron7 Donor GC→TA Lung (NSCLC) ca Intron 7 Accept GC→CG Lung (SCLC) caIntron 7 Accept CG→AT AML Intron 7 Donor GC→TA Lung (SCLC) ca Intron 9Donor GC→TA Lung (SCLC) ca

A. CFLP Analysis of p53 Mutations in Clinical Samples

To permit the identification of mutations in the p53 gene from clinicalsamples, nucleic acid comprising p53 gene sequences are prepared. Thenucleic acid may comprise genomic DNA, RNA or cDNA forms of the p53gene. Nucleic acid may be extracted from a variety of clinical samples(fresh or frozen tissue, suspensions of cells (e.g., blood), cerebralspinal fluid, sputum, etc.) using a variety of standard techniques orcommerically available kits. For example, kits which allow the isolationof RNA or DNA from tissue samples are available from Qiagen, Inc.(Chatsworth, Calif.) and Stratagene (LaJolla, Calif.), respectively.Total RNA may be isolated from tissues and tumors by a number of methodsknown to those skilled in the art and commercial kits are available tofacilitate the isolation. For example, the RNEASY kit (Qiagen Inc.,Chatsworth, Calif.) provides protocol, reagents and plasticware topermit the isolation of total RNA from tissues, cultured cells orbacteria, with no modification to the manufacturer's instructions, inapproximately 20 minutes. Should it be desirable, in the case ofeukaryotic RNA isolates, to further enrich for messenger RNAs, thepolyadenylated RNAs in the mixture may be specifically isolated bybinding to an oligo-deoxythymidine matrix, through the use of a kit suchas the OLIGOTEX kit (Qiagen). Comparable isolation kits for both ofthese steps are available through a number of commercial suppliers.

In addition, RNA may be extracted from samples, including biopsyspecimens, conveniently by lysing the homogenized tissue in a buffercontaining 0.22 M NaCl, 0.75 mM MgCl₂, 0.1 M Tris-HCl, pH 8.0, 12.5 mMEDTA, 0.25% NP40, 1% SDS, 0.5 mM DTT, 500 u/ml placental RNAse inhibitorand 200 μg/ml Proteinase K. Following incubation at 37° C. for 30 min,the RNA is extracted with phenol:chloroform (1:1) and the RNA isrecovered by ethanol precipitation.

Since the majority of p53 mutations are found within exons 5-8, it isconvenient as a first analysis to examine a PCR fragment spanning thisregion. PCR fragments spanning exons 5-8 may be amplified from clinicalsamples using the technique of RT-PCR (reverse transcription-PCR); kitswhich permit the user to start with tissue and produce a PCR product areavailable from Perkin Elmer (Norwalk, Conn.) and Stratagene (LaJolla,Calif.). The RT-PCR technique generates a single-stranded cDNAcorresponding to a chosen segment of the coding region of a gene byusing reverse transcription of RNA; the single-stranded cDNA is thenused as template in the PCR. In the case of the p53 gene, anapproximately 600 by fragment spanning exons 5-8 is generated usingprimers located in the coding region immediately adjacent to exons 5 and8 in the RT-PCR. The PCR amplified segment is then subjected to the CFLPreaction and the reaction products are analysed as described above insection VIII.

Fragments suitable for CFLP analysis may also be generated by PCRamplification of genomic DNA. DNA is extracted from a sample and primerscorresponding to sequences present in introns 4 and 8 are used toamplify a segment of the p53 gene spanning exons 5-8 which includesintrons 5-7 (an approximately 2 kb fragment). If it is desirable to usesmaller fragments of DNA in the CFLP reaction, primers may be chosen toamplify smaller (1 kb or less) segments of genomic DNA or alternativelya large PCR fragment may be divided into two or more smaller fragmentsusing restriction enzymes.

In order to facilitate the identification of p53 mutations in theclinical setting, a library containing the CFLP pattern produced bypreviously characterized mutations may be provided. Comparison of thepattern generated using nucleic acid derived from a clinical sample withthe patterns produced by cleavage of known and characterized p53mutations will allow the rapid identification of the specific p53mutation present in the patient's tissue. The comparison of CFLPpatterns from clinical samples to the patterns present in the librarymay be accomplished by a variety of means. The simplest and leastexpensive comparison involves visual comparison. Given the large numberof unique mutations known at the p53 locus, visual (i.e., manual)comparison may be too time-consuming, especially when large numbers ofclinical isolates are to be screened. Therefore the CFLP patterns or“bar codes” may be provided in an electronic format for ease andefficiency in comparison. Electronic entry may comprise storage of scansof gels containing the CFLP products of the reference p53 mutations(using for example, the GENEREADER and GEL DOCTOR Fluorescence Geldocumentation system (BioRad, Hercules, Calif.) or the IMAGE MASTER(Pharmacia Biotech, Piscataway, N.J.). Alternatively, as the detectionof cleavage patterns may be automated using DNA sequencinginstrumentation (see Example 20), the banding pattern may be stored asthe signal collected from the appropriate channels during an automatedrun (examples of instrumentation suitable for such analysis and datacollection include fluorescence-based gel imagers such as fluoroimagersproduced by Molecular Dynamics and Hitachi or by real-timeelectrophoresis detection systems such as the ABI 377 or Pharmacia ALFDNA Sequencer).

B. Generation of a Library of Characterized p53 Mutations

The generation of a library of characterized mutations will enableclinical samples to be rapidly and directly screened for the presence ofthe most common p53 mutations. Comparison of CFLP patterns generatedfrom clinical samples to the p53 bar code library will establish boththe presence of a mutation in the p53 gene and its precise identitywithout the necessity of costly and time consuming DNA sequenceanalysis.

The p53 bar code library is generated using reverse genetics.Engineering of p53 mutations ensures the identity and purity of each ofthe mutations as each engineered mutation is confirmed by DNAsequencing. The individual p53 mutations in p53 bar code library aregenerated using the 2-step “recombinant PCR” technique (Higuchi, R.(1991) In Ehrlich, H. A. (Ed.), PCR Technology: Principles andApplications for DNA Amplification, Stockton Press, New York, pp. 61-70and Nelson, R. M. and Long, G. L. (1989) Analytical Biochem. 180:147).FIG. 77 provides a schematic representation of one method of a 2-steprecombinant PCR technique that may be used for the generation of p53mutations.

The template for the PCR amplifications is the entire human p53 cDNAgene. In the first of the two PCRs (designated “PCR 1” in FIG. 77), anoligonucleotide containing the engineered mutation (“oligo A” in FIG.77) and an oligonucleotide containing a 5′ arm of approximately 20non-complementary bases (“oligo B”) are used to amplify a relativelysmall region of the target DNA (100-200 bp). The resulting amplificationproduct will contain the mutation at its extreme 5′ end and a foreignsequence at its 3′ end. The 3′ sequence is designed to include a uniquerestriction site (e.g., Eco RI) to aid in the directional cloning of thefinal amplification fragment (important for purposes of sequencing andarchiving the DNA containing the mutation). The product generated in theupstream or first PCR may be gel purified if desired prior to the use ofthis first PCR product in the second PCR; however gel purification isnot required once it is established that this fragment is the onlyspecies amplified in the PCR.

The small PCR fragment containing the engineered mutation is then usedto direct a second round of PCR (PCR 2). In PCR 2, the target DNA is alarger fragment (approximately 1 kb) of the same subcloned region of thep53 cDNA. Because the sequence at the 3′ end of the small PCR fragmentis not complementary to any of the sequences present in the target DNA,only that strand in which the mismatch is at the extreme 5′ end isamplified in PCR 2 (a 3′ non-templated arm cannot be extended in PCR).Amplification is accomplished by the addition of a primer complementaryto a region of the target DNA upstream of the locus of the engineeredmutation (“oligo C”) and by the addition of a primer complementary tothe 5′ noncomplementary sequence of the small product of PCR 1 (“oligoD”). By directing amplification from the noncomplementary sequence, thisprocedure results in the specific amplification of only those sequencescontaining the mutation. In order to facilitate cloning of these PCRproducts into a standard vector, a second unique restriction site can beengineered into oligo C (e.g., HindIII).

The use of this 2-step PCR approach requires that only one primer besynthesized for each mutant to be generated after the initial set-up ofthe system (i.e., oligo A). Oligos B, C and D can be used for allmutations generated within a given region. Because oligos C and D aredesigned to include different and unique restriction sites, subsequentdirectional cloning of these PCR products into plasmid vectors (such aspUC 19) is greatly simplified. Selective amplification of only thosesequences that include the desired mutational change simplifiesidentification of mutation-containing clones as only verification of thesequence of insert containing plasmids is required. Once the sequence ofthe insert has been verified, each mutation-containing clone may bemaintained indefinitely as a bacterial master stock. In addition, DNAstocks of each mutant can be maintained in the form of large scale PCRpreparations. This permits distribution of either bactera harboringplasmids containing a given mutation or a PCR preparation to bedistributed as individual controls in kits containing reagents for thescanning of p53 mutations in clinical samples or as part of asupplemental master p53 mutation library control kit.

An alternative 2-step recombinant PCR is diagrammed in FIG. 78, anddescribed in Example 32. In this method two mutagenic oligonucleotides,one for each strand, are synthesized. These oligonucleotides aresubstantially complementary to each other but are opposite inorientation. That is, one is positioned to allow amplification of an“upstream” region of the DNA, with the mutation incorporated into the 3′proximal region of the upper, or sense strand, while the other ispositioned to allow amplification of a “downstream” segment with theintended mutation incorporated into the 5′ proximal region of the upper,or sense strand. These two double stranded products share the sequenceprovided by these mutagenic oligonucleotides. When purified, combined,denatured and annealed, those strands that anneal with recessed 3′ endscan be extended or filled in by the action of DNA polymerase, thusrecreating a full length molecules with the mutation in the centralregion. This recombinant can be amplified by the use of the “outer”primer pair, those used to make the 5′ end of the “upstream” and the 3′end of the “downstream” intermediate fragments.

While extra care must be taken with this method (in comparison with themethod described above) because the outer primers can amplify both therecombinant and the un-modified sequence, this method does allow rapidrecombinant PCR to be performed using existing end primers, and withoutthe introduction of foreign sequences. In summary, this method is oftenused if only a few recombinations are to be performed. When largevolumes of mutagenic PCRs are to be performed, the first describedmethod is preferable as the first method requires a single oligo besynthesized for each mutagenesis and only recombinants are amplified.

An important feature of kits designed for the identification of p53mutations in clinical samples is the inclusion of the specific primersto be used for generating PCR fragments to be analyzed for CFLPanalysis. While DNA fragments from 100 to over 1500 by can bereproducibly and accurately analyzed for the presence of sequencepolymorphisms by this technique, the precise patterns generated fromdifferent length fragments of the same input DNA sequence will of coursevary. Not only are patterns shifted relative to one another depending onthe length of the input DNA, but in some cases, more long rangeinteractions between distant regions of long DNA fragments may result inthe generation of additional cleavage products not seen with shorterinput DNA products. For this reason, exact matches with the bar codelibrary will be assured through the use of primers designed to amplifythe same size fragment from the clinical samples as was used to generatea given version of the p53 bar code library.

C. Detection of Unique CFLP Patterns for p53 Mutations

The simplest and most direct method of analyzing the DNA fragmentsproduced in the CFLP reaction is by gel electrophoresis. Becauseelectrophoresis is widely practiced and easily accessible, initialefforts have been aimed at generating a database in this familiarformat. It should, however, be noted that resolution of DNA fragmentsgenerated by CFLP analysis is not limited to electrophoretic methods.Mass spectrometry, chromotography, fluorescence polarization, and chiphybridization are all approaches that are currently being refined anddeveloped in a number of research laboratories. Once generated, the CFLPdatabase is easily adapted to analysis by any of these methods.

There are several possible alternatives available for detection of CFLPpatterns. A critical user benefit of CFLP analysis is that the resultsare not dependent on the chosen method of DNA detection. DNA fragmentsmay be labeled with a radioisotope (e.g., a ³²P or ³⁵S-labelednucleotide) placed at either the 5′ or 3′ end of the nucleic acid oralternatively the label may be distributed throughout the nucleic acid(i.e., an internally labeled substrate). The label may be a nonisotopicdetectable moiety, such as a fluorophore which can be detected directly,or a reactive group which permits specific recognition by a secondaryagent. CFLP patterns have been detected by immunostaining, biotin-avidininteractions, autoradiography and direct fluorescence imaging. Sinceradiation use is in rapid decline in clinical settings and since bothimmunostaining and biotin-avidin based detection schemes requiretime-consuming transfer of DNA onto an expensive membrane support,fluorescence-based detection methods may be preferred. It is importantto note, however, that any of the above methods may be used to generateCFLP bar codes to be input into the database.

In addition to their being a direct, non-isotopic means of detectingCFLP patterns, fluorescence-based schemes offer a noteworthy additionaladvantage in clinical applications. CFLP analysis allows the analysis ofseveral samples in the same tube and in the same lane on a gel. This“multiplexing” permits rapid and automated comparison of a large numberof samples in a fraction of the time and for a lower cost than can berealized through individual analysis of each sample. This approach opensthe door to several alternative applications. A researcher could decideto double, triple or quadruple (up to 4 dyes have been demonstrated tobe detectable and compatible in a single lane in commerically availableDNA sequencing instrumentation such as the ABI 373/377) the number ofsamples run on a given gel. Alternatively, the analyst may include anormal p53 gene sample in each tube, and each gel lane, along with adifferentially labeled size standard, as a internal standard to verifyboth the presence and the exact location(s) of a pattern difference(s)between the normal p53 gene and putative mutants.

IV. Detection and Identification of Pathogens Using the CFLP Method

A. Detection and Identification of Hepatitis C Virus

Hepatitis C virus (HCV) infection is the predominant cause ofpost-transfusion non-A, non-B (NANB) hepatitis around the world. Inaddition, HCV is the major etiologic agent of hepatocellular carcinoma(HCC) and chronic liver disease world wide. HCV infection is transmittedprimarily to blood transfusion recipients and intravenous drug usersalthough maternal transmission to offspring and transmission torecipients of organ transplants have been reported.

The genome of the positive-stranded RNA hepatitis C virus comprisesseveral regions including 5′ and 3′ noncoding regions (i.e., 5′ and 3′untranslated regions) and a polyprotein coding region which encodes thecore protein (C), two envelope glycoproteins (E1 and E2/NS1) and sixnonstructural glycoproteins (NS2—NS5b). Molecular biological analysis ofthe small (9.4 kb) RNA genome has showed that some regions of the genomeare very highly conserved between isolates, while other regions arefairly rapidly changeable. The 5′ noncoding region (NCR) is the mosthighly conserved region in the HCV. These analyses have allowed theseviruses to be divided into six basic genotype groups, and then furtherclassified into over a dozen sub-types (the nomenclature and division ofHCV genotypes is evolving; see Altamirano et al., J. Infect. Dis.171:1034 (1995) for a recent classification scheme). These viral groupsare associated with different geographical areas, and accurateidentification of the agent in outbreaks is important in monitoring thedisease. While only Group 1 HCV has been observed in the United States,multiple HCV genotypes have been observed in both Europe and Japan.

The ability to determine the genotype of viral isolates also allowscomparisons of the clinical outcomes from infection by the differenttypes of HCV, and from infection by multiple types in a singleindividual. HCV type has also been associated with differential efficacyof treatment with interferon, with Group 1 infected individuals showinglittle response (Kanai et al., Lancet 339:1543 (1992) and Yoshioka etal., Hepatology 16:293 (1992)). Pre-screening of infected individualsfor the viral type will allow the clinician to make a more accuratediagnosis, and to avoid costly but fruitless drug treatment.

Existing methods for determining the genotype of HCV isolates includePCR amplification of segments of the HCV genome coupled with either DNAsequencing or hybridization to HCV-specific probes, RFLP analysis of PCRamplified HCV DNA anything else?. All of these methods suffer from thelimitations discussed above (i.e., DNA sequencing is too labor-intensiveand expensive to be practical in clinical laboratory settings; RFLPanalysis suffers from low sensitivity).

Universal and genotype specific primers have been designed for theamplification of HCV sequences from RNA extracted from plasma or serum(Okamoto et al. J. Gen. Virol. 73:673 (1992); Yoshioka et al.,Hepatology 16:293 (1992) and Altamirano et al., supra). These primerscan be used to generate PCR products which serve as substrates in theCFLP assay of the present invention. As shown herein CFLP analysisprovides a rapid and accurate method of typing HCV isolates. CFLPanalysis of HCV substrates allows a distinction to be made between themajor genotypes and subtypes of HCV thus providing improved methods forthe genotyping of HCV isolates.

B. Detection and Identification of Multi-Drug Resistant M. tuberculosis

In the past decade there has been a tremendous resurgence in theincidence of tuberculosis in this country and throughout the world. Inthe United States, the incidence of tuberculosis has risen steadilyduring past decade, accounting for 2000 deaths annually, with as many as10 million Americans infected with the disease. The situation iscritical in New York City, where the incidence has more than doubled inthe past decade, accounting for 14% of all new cases in the UnitedStates in 1990 (Frieden et al., New Engl. J. Med. 328:521 (1993)).

The crisis in New York City is particularly dire because a significantproportion (as many as one-third) of the recent cases are resistant toone or more antituberculosis drugs (Frieden et al, supra and Hughes,Scrip Magazine May (1994)). Multi-drug resistant tuberculosis (MDR-TB)is an iatrogenic disease that arises from incomplete treatment of aprimary infection (Jacobs, Jr., Clin. Infect. Dis. 19:1 (1994)). MDR-TBappears to pose an especially serious risk to the immunocompromised, whoare more likely to be infected with MDR-TB strains than are otherwisehealthy individuals (Jacobs, Jr., supra). The mortality rate of MDR-TBin immunocompromised individuals is alarmingly high, often exceeding90%, compared to a mortality rate of <50% in otherwise uncompromisedindividuals (Donnabella et al., Am. J. Respir. Dis. 11:639 (1994)).

From a clinical standpoint, tuberculosis has always been difficult todiagnose because of the extremely long generation time of Mycobacteriumtuberculosis as well as the environmental prevalence of other, fastergrowing mycobacterial species. The doubling time of M tuberculosis is20-24 hours, and growth by conventional methods typically requires 4 to6 weeks to positively identify M. tuberculosis (Jacobs, Jr. et al.,Science 260:819 (1993) and Shinnick and Jones in Tuberculosis:Pathogenesis, Protection and Control, Bloom, ed., American Society ofMicrobiology, Washington, D.C. (1994), pp. 517-530). It can take anadditional 3 to 6 weeks to diagnose the drug susceptibility of a givenstrain (Shinnick and Jones, supra). Needless to say, the health risks tothe infected individual, as well as to the public, during a protractedperiod in which the patient may or may not be symptomatic, but is almostcertainly contagious, are considerable. Once a drug resistance profilehas been elucidated and a diagnosis made, treatment of a single patientcan cost up to $250,000 and require 24 months.

The recent explosion in the incidence of the disease, together with thedire risks posed by MDR strains, have combined to spur a burst ofresearch activity and commercial development of procedures and productsaimed at accelerating the detection of M. tuberculosis as well theelucidation of drug resistance profiles of M. tuberculosis clinicalisolates. A number of these methods are devoted primarily to the task ofdetermining whether a given strain is M. tuberculosis or a mycobacterialspecies other than tuberculosis. Both culture based methods andnucleic-acid based methods have been developed that allow M.tuberculosis to be positively identified more rapidly than by classicalmethods: detection times have been reduced from greater than 6 weeks toas little as two weeks (culture-based methods) or two days (nucleicacid-based methods). While culture-based methods are currently inwide-spread use in clinical laboratories, a number of rapid nucleicacid-based methods that can be applied directly to clinical samples areunder development. For all of the techniques described below, it isnecessary to first “decontaminate” the clinical samples, such as sputum(usually done by pretreatment with N-acetyl L-cysteine and NaOH) toreduce contamination by non-mycobacterial species (Shinnick and Jones,supra.)

The polymerase chain reaction (PCR) has been applied to the detection ofM. tuberculosis and can be used to detect its presence directly fromclinical specimens within one to two days. The more sensitive techniquesrely on a two-step procedure: the first step is the PCR amplificationitself, the second is an analytical step such as hybridization of theamplicon to a M. tuberculosis-specific oligonucleotide probe, oranalysis by RFLP or DNA sequencing (Shinnick and Jones, supra).

The Amplified M. tuberculosis Direct Test (AMTDT; Gen-Probe) relies onTranscription Mediated Amplification (TMA; essentially a self-sustainedsequence reaction (3SR) amplification) to amplify target rRNA sequencesdirectly from clinical specimens. Once the rRNA has been amplified, itis then detected by a dye-labeled assay such as the PACE2. This assay ishighly subject to inhibition by substances present in clinical samples.

The Cycling Probe Reaction (CPR; ID Biomedical). This technique, whichis under development as a diagnostic tool for detecting the presence ofM. tuberculosis, measures the accumulation of signal probe molecules.The signal amplification is accomplished by hybridizing tripartiteDNA-RNA-DNA probes to target nucleic acids, such as M.tuberculosis-specific sequences. Upon the addition of RNAse H, the RNAportion of the chimeric probe is degraded, releasing the DNA portions,which accumulate linearly over time to indicate that the target sequenceis present (Yule, Bio/Technology 12:1335 (1994)). The need to use of RNAprobes is a drawback, particularly for use in crude clinical samples,where RNase contamination is often rampant.

The above nucleic acid-based detection and differentiation methods offera clear time savings over the more traditional, culture-based methods.While they are beginning to enter the clinical setting, their usefulnessin the routine diagnosis of M. tuberculosis is still in question, inlarge part because of problems with associated with cross-contaminationand low-sensitivity relative to culture-based methods. In addition, manyof these procedures are limited to analysis of respiratory specimens(Yule, Bio/Technology 12:1335 (1994)).

ii) Determination of the antibiotic resistance profile of M.tuberculosis

a) Culture-based methods: Once a positive identification of M.tuberculosis has been made, it is necessary to characterize the extentand nature of the strain's resistance to antibiotics. The traditionalmethod used to determine antibiotic resistance is the direct proportionagar dilution method, in which dilutions of culture are plated on mediacontaining antibiotics and on control media without antibiotics. Thismethod typically adds an additional 2-6 weeks to the time required fordiagnosis and characterization of an unknown clinical sample (Jacobs,Jr., supra).

The Luciferase Reporter Mycobacteriophage (LRM) assay was firstdescribed in 1993 (Jacobs, Jr. et al., Science 260:819 (1993)). In thisassay, a mycobacteriophage containing a cloned copy of the luciferasegene is used to infect mycobacterial cultures. In the presence ofluciferin and ATP, the expressed luciferase produces photons, easilydistinguishable by eye or by a luminometer, allowing a precisedetermination of the extent of mycobacterial growth in the presence ofantibiotics. Once sufficient culture has been obtained (usually 10-14days post-inoculation), the assay can be completed in 2 days. Thismethod suffers from the fact that the LRM are not specific for M.tuberculosis: they also infect M. smegmatis and M. bovis (e.g., BCG),thereby complicating the interpretation of positive results.Discrimination between the two species must be accomplished by growth onspecialized media which does not support the growth of M. tuberculosis(e.g., NAP media). This confirmation requires another 2 to 4 days.

The above culture-based methods for determining antibiotic resistancewill continue to play a role in assessing the effectiveness of putativenew anti-mycobacterial agents and those drugs for which a genetic targethas not yet been identified. However, recent success in elucidating themolecular basis for resistance to a number of anti-mycobacterial agents,including many of the front-line drugs, has made possible the use ofmuch faster, more accurate and more informative DNA polymorphism-basedassays.

b) DNA-based methods: Genetic loci involved in resistance to isoniazid,rifampin, streptomycin, fluoroquinolones, and ethionamide have beenidentified (Jacobs, Jr., supra; Heym et al., Lancet 344:293 (1994) andMorris et al., J. Infect. Dis. 171:954 (1995)). A combination ofisoniazid (inh) and rifampin (rif) along with pyrazinamide andethambutol or streptomycin, is routinely used as the first line ofattack against confirmed cases of M. tuberculosis (Banerjee et al.,Science 263:227 (1994)). Consequently, resistance to one or more ofthese drugs can have disastrous implications for short coursechemotherapy treatment. The increasing incidence of such resistantstrains necessitates the development of rapid assays to detect them andthereby reduce the expense and community health hazards of pursuingineffective, and possibly detrimental, treatments. The identification ofsome of the genetic loci involved in drug resistance has facilitated theadoption of mutation detection technologies for rapid screening ofnucleotide changes that result in drug resistance. The availability ofamplification procedures such as PCR and SDA, which have been successfulin replicating large amounts of target DNA directly from clinicalspecimens, makes DNA-based approaches to antibiotic profiling far morerapid than conventional, culture-based methods.

The most widely employed techniques in the genetic identification ofmutations leading to drug resistance are DNA sequencing, RestrictionFragment Length Polymorphism (RFLP), PCR-Single Stranded ConformationalPolymorphism (PCR-SSCP), and PCR-dideoxyfingerprinting (PCR-ddF). All ofthese techniques have drawbacks as discussed above. None of them offersa rapid, reproducible means of precisely and uniquely identifyingindividual alleles.

In contrast the CFLP method of the present invention provides anapproach that relies on structure specific cleavage to generate distinctcollections of DNA fragments. This method is highly sensitive (>98%) inits ability to detect sequence polymorphisms, and requires a fraction ofthe time, skill and expense of the techniques described above.

The application of the CFLP method to the detection of MDR-TB isillustrated herein using segments of DNA amplified from the rpoB andkatG genes. Other genes associated with MDR-TB, including but notlimited to those involved in conferring resistance to isoniazid (inhA),streptomycin (rpsL and rrs), and fluoroquinoline (gyrA), are equallywell suited to the CFLP assay.

C. Detection and Identification of Bacterial Pathogens

Identification and typing of bacterial pathogens is critical in theclinical management of infectious diseases. Precise identity of amicrobe is used not only to differentiate a disease state from a healthystate, but is also fundamental to determining whether and whichantibiotics or other antimicrobial therapies are most suitable fortreatment. Traditional methods of pathogen typing have used a variety ofphenotypic features, including growth characteristics, color, cell orcolony morphology, antibiotic susceptibility, staining, smell andreactivity with specific antibodies to identify bacteria. All of thesemethods require culture of the suspected pathogen, which suffers from anumber of serious shortcomings, including high material and labor costs,danger of worker exposure, false positives due to mishandling and falsenegatives due to low numbers of viable cells or due to the fastidiousculture requirements of many pathogens. In addition, culture methodsrequire a relatively long time to achieve diagnosis, and because of thepotentially life-threatening nature of such infections, antimicrobialtherapy is often started before the results can be obtained. In manycases the pathogens are very similar to the organisms that make up thenormal flora, and may be indistinguishable from the innocuous strains bythe methods cited above. In these cases, determinion of the presence ofthe pathogenic strain may require the higher resolution afforded by morerecently developed molecular typing methods.

A number of methods of examining the genetic material from organisms ofinterest have been developed. One way of performing this type ofanalysis is by hybridization of species-specific nucleic acid probes tothe DNA or RNA from the organism to be tested. This may be done byimmobilizing the denatured nucleic acid to be tested on a membranesupport, and probing with labeled nucleic acids that will bind only inthe presence of the DNA or RNA from the pathogen. In this way, pathogenscan be identified. Organisms can be further differentiated by using theRFLP method described above, in which the genomic DNA is digested withone or more restriction enzymes before electrophoretic separation andtransfer to a nitrocellulose or nylon membrane support. Probing with thespecies-specific nucleic acid probes will reveal a banding pattern that,if it shows variation between isolates, can be used as a reproducibleway of discriminating between strains. However, these methods aresusceptible to the drawbacks outlined above: hybridization-based assaysare time-consuming and may give false or misleading results if thestringency of the hybridization is not well controlled, and RFLPidentification is dependent on the presence of suitable restrictionsites in the DNA to be analyzed.

To address these concerns about hybridization and RFLP as diagnostictools, several methods of molecular analysis based on polymerase chainreaction (PCR) amplification have gained popularity. In onewell-accepted method, called PCR fingerprinting, the size of a fragmentgenerated by PCR is used as an identifier. In this type of assay, theprimers are targeted to regions containing variable numbers of tandemrepeated sequences (referred to as VNTRs an eukaryotes). The number ofrepeats, and thus the length of the PCR amplicon, can be characteristicof a given pathogen, and co-amplification of several of these loci in asingle reaction can create specific and reproducible fingerprints,allowing discrimination between closely related species.

In some cases where organisms are very closely related, however, thetarget of the amplification does not display a size difference, and theamplified segment must be further probed to achieve more preciseidentification. This may be done on a solid support, in a fashionanalogous to the whole-genome hybridization described above, but thishas the same problem with variable stringency as that assay.Alternatively, the interior of the PCR fragment may be used as atemplate for a sequence-specific ligation event. As outlined above forthe LCR, in this method, single stranded probes to be ligated arepositioned along the sequence of interest on either side of anidentifying polymorphism, so that the success or failure of the ligationwill indicate the presence or absence of a specifice nucleotide sequenceat that site. With either hybridization or ligation methods of PCRproduct analysis, knowledge of the precise sequence in the area of probebinding must be obtained in advance, and differences outside the probebinding area are not detected. These methods are poorly suited to theexamination and typing of new isolates that have not been fullycharacterized.

In the methods of the present invention, primers that recognizeconserved regions of bacterial ribosomal RNA genes allow amplificationof segments of these genes that include sites of variation. Thevariations in ribosomal gene sequences have become an accepted methodnot only of differentiating between similar organisms on a DNA sequencelevel, but their consistent rate of change allows these sequences to beused to evaluate the evolutionary relatedness of organisms. That is tosay, the more similar the nucleic acid is at the sequence level, themore closely related the organisms in discussion are considered to be.(Woese, Bacterial Evolution. Microbiological Reviews, vol 51, No. 2.1987). The present invention allows the amplification products derivedfrom these sequences to be used to create highly individual barcodes(i.e., cleavage patterns), allowing the detection of sequencepolymorphisms without prior knowledge of the site, character or even thepresence of said polymorphisms. With appropriate selection of primers,amplification can be made to be either all-inclusive (e.g., using themost highly conserved ribosomal sequences) to allow comparison ofdistantly related organisms, or the primers can be chosen to be veryspecific for a given genus, to allow examination at the species andsubspecies level. While the examination of ribosomal genes is extremelyuseful in these characterizations, the use of the CFLP method inbacterial typing is not limited to these genes. Other genes, includingbut not limited to those associated with specific growthcharacteristics, (e.g., carbon source preference, antibiotic resistance,resistance to methycillin or antigen production), or with particularcell morphologies (such as pilus formation) are equally well suited tothe CFLP assay.

D. Extraction of Nucleic Acids From Clinical Samples

To provide nucleic acid substrates for use in the detection andidentification of microorganisms in clinical samples using the CFLPassay, nucleic acid is extracted from the sample. The nucleic acid maybe extracted from a variety of clinical samples (fresh or frozen tissue,suspensions of cells (e.g., blood), cerebral spinal fluid, sputum,urine, etc.) using a variety of standard techniques or commerciallyavailable kits. For example, kits which allow the isolation of RNA orDNA from tissue samples are available from Qiagen, Inc. (Chatsworth,Calif.) and Stratagene (LaJolla, Calif.). For example, the QIAAMP Bloodkits permit the isolation of DNA from blood (fresh, frozen or dried) aswell as bone marrow, body fluids or cell suspensions. QIAAMP tissue kitspermit the isolation of DNA from tissues such as muscles, organs andtumors.

It has been found that crude extracts from relatively homogenousspecimens (such as blood, bacterial colonies, viral plaques, or cerebralspinal fluid) are better suited to severing as templates for theamplification of unique PCR products than are more composite specimens(such as urine, sputum or feces;) (Shibata in PCR: The Polymerase ChainReaction, Mullis et al., eds., Birkhauser, Boston (1994), pp. 47-54).Samples which contain relatively few copies of the material to beamplified (i.e., the target nucleic acid), such as cerebral spinalfluid, can be added directly to a PCR. Blood samples have posed aspecial problem in PCRs due to the inhibitory properties of red bloodcells. The red blood cells must be removed prior to the use of blood ina PCR; there are both classical and commercially available methods forthis purpose (e.g., QIAAMP Blood kits, passage through a CHELEX 100column (BioRad), etc.). Extraction of nucleic acid from sputum, thespecimen of choice for the direct detection of M. tuberculosis, requiresprior decontamination to kill or inhibit the growth of other bacterialspecies. This decontamination is typically accomplished by treatment ofthe sample with N-acetyl L-cysteine and NaOH (Shinnick and Jones,supra). This decontamination process is necessary only when the sputumspecimen is to be cultured prior to analysis.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the disclosure which follows, the following abbreviations apply: ° C.(degrees Centigrade); g (gravitational field); vol (volume); w/v (weightto volume); v/v (volume to volume); BSA (bovine serum albumin); CTAB(cetyltrimethylammonium bromide); HPLC (high pressure liquidchromatography); DNA (deoxyribonucleic acid); IVS (interveningsequence); p (plasmid); μl (microliters); ml (milliliters); μg(micrograms); pmoles (picomoles); mg (milligrams); MOPS(3-(N-Morpholino)propanesulfonic acid); M (molar); mM (milliMolar); μM(microMolar); nm (nanometers); kdal (kilodaltons); OD (optical density);EDTA (ethylene diamine tetra-acetic acid); FITC (fluoresceinisothiocyanate); SDS (sodium dodecyl sulfate); NaPO₄ (sodium phosphate);Tris (tris(hydroxymethyl)-aminomethane); PMSF(phenylmethylsulfonylfluoride); TBE (Tris-Borate-EDTA, i.e., Tris buffertitrated with boric acid rather than HCl and containing EDTA); PBS(phosphate buffered saline); PPBS (phosphate buffered saline containing1 mM PMSF); PAGE (polyacrylamide gel electrophoresis); TWEEN(polyoxyethylene-sorbitan); Boehringer Mannheim (Boehringer Mannheim,Indianapolis, Ind.); Dynal (Dynal A. S., Oslo, Norway); Epicentre(Epicentre Technologies, Madison, Wis.); National Biosciences (NationalBiosciences, Plymouth, Minn.); New England Biolabs (New England Biolabs,Beverly, Mass.); Novagen (Novagen, Inc., Madison, Wis.); Perkin Elmer(Perkin Elmer, Norwalk, Conn.); Promega Corp. (Promega Corp., Madison,Wis.); RJ Research (RJ Research, Inc., Watertown, Mass.); Stratagene(Stratagene Cloning Systems, La Jolla, Calif.); USB (U.S. Biochemical,Cleveland, Ohio).

Example 1 Characteristics of Native Thermostable DNA Polymerases

A. 5′ Nuclease Activity Of DNAPTaq

During the polymerase chain reaction (PCR) (Saiki et al., Science239:487 (1988); Mullis and Faloona, Methods in Enzymology 155:335(1987)), DNAPTaq is able to amplify many, but not all, DNA sequences.One sequence that cannot be amplified using DNAPTaq is shown in FIG. 6(Hairpin structure is SEQ ID NO:15, PRIMERS are SEQ ID NOS:16-17.) ThisDNA sequence has the distinguishing characteristic of being able to foldon itself to form a hairpin with two single-stranded arms, whichcorrespond to the primers used in PCR.

To test whether this failure to amplify is due to the 5′ nucleaseactivity of the enzyme, we compared the abilities of DNAPTaq and DNAPStfto amplify this DNA sequence during 30 cycles of PCR. Syntheticoligonucleotides were obtained from The Biotechnology Center at theUniversity of Wisconsin-Madison. The DNAPTaq and DNAPStf were fromPerkin Elmer (i.e., AMPLITAQ DNA polymerase and the Stoffel fragment ofAMPLITAQ DNA polymerase). The substrate DNA comprised the hairpinstructure shown in FIG. 6 cloned in a double-stranded form into pUC19.The primers used in the amplification are listed as SEQ ID NOS:16-17.Primer SEQ ID NO:17 is shown annealed to the 3′ arm of the hairpinstructure in FIG. 6. Primer SEQ ID NO:16 is shown as the first 20nucleotides in bold on the 5′ arm of the hairpin in FIG. 6.

Polymerase chain reactions comprised 1 ng of supercoiled plasmid targetDNA, 5 pmoles of each primer, 40 μM each dNTP, and 2.5 units of DNAPTaqor DNAPStf, in a 50 μl solution of 10 mM Tris.Cl pH 8.3. The DNAPTaqreactions included 50 mM KCl and 1.5 mM MgCl₂. The temperature profilewas 95° C. for 30 sec., 55° C. for 1 min. and 72° C. for 1 min., through30 cycles. Ten percent of each reaction was analyzed by gelelectrophoresis through 6% polyacrylamide (cross-linked 29:1) in abuffer of 45 mM Tris.Borate, pH 8.3, 1.4 mM EDTA.

The results are shown in FIG. 7. The expected product was made byDNAPStf (indicated simply as “S”) but not by DNAPTaq (indicated as “T”).We conclude that the 5′ nuclease activity of DNAPTaq is responsible forthe lack of amplification of this DNA sequence.

To test whether the 5′ unpaired nucleotides in the substrate region ofthis structured DNA are removed by DNAPTaq, the fate of the end-labeled5′ arm during four cycles of PCR was compared using the same twopolymerases (FIG. 8). The hairpin templates, such as the one describedin FIG. 6, were made using DNAPStf and a ³²P-5′-end-labeled primer. The5′-end of the DNA was released as a few large fragments by DNAPTaq butnot by DNAPStf. The sizes of these fragments (based on their mobilities)show that they contain most or all of the unpaired 5′ arm of the DNA.Thus, cleavage occurs at or near the base of the bifurcated duplex.These released fragments terminate with 3′ OH groups, as evidenced bydirect sequence analysis, and the abilities of the fragments to beextended by terminal deoxynucleotidyl transferase.

FIGS. 9-11 show the results of experiments designed to characterize thecleavage reaction catalyzed by DNAPTaq. Unless otherwise specified, thecleavage reactions comprised 0.01 pmoles of heat-denatured, end-labeledhairpin DNA (with the unlabeled complementary strand also present), 1pmole primer (complementary to the 3′ arm) and 0.5 units of DNAPTaq(estimated to be 0.026 pmoles) in a total volume of 10 μl of 10 mMTris-Cl, ph 8.5, 50 mM KCl and 1.5 mM MgCl₂. As indicated, somereactions had different concentrations of KCl, and the precise times andtemperatures used in each experiment are indicated in the individualfigures. The reactions that included a primer used the one shown in FIG.6 (SEQ ID NO:17). In some instances, the primer was extended to thejunction site by providing polymerase and selected nucleotides.

Reactions were initiated at the final reaction temperature by theaddition of either the MgCl₂ or enzyme. Reactions were stopped at theirincubation temperatures by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. The T_(m) calculations listed were madeusing the OLIGO primer analysis software from National Biosciences, Inc.These were determined using 0.25 μM as the DNA concentration, at either15 or 65 mM total salt (the 1.5 mM MgCl₂ in all reactions was given thevalue of 15 mM salt for these calculations).

FIG. 9 is an autoradiogram containing the results of a set ofexperiments and conditions on the cleavage site. FIG. 9A is adetermination of reaction components that enable cleavage. Incubation of5′-end-labeled hairpin DNA was for 30 minutes at 55° C., with theindicated components. The products were resolved by denaturingpolyacrylamide gel electrophoresis and the lengths of the products, innucleotides, are indicated. FIG. 9B describes the effect of temperatureon the site of cleavage in the absence of added primer. Reactions wereincubated in the absence of KCl for 10 minutes at the indicatedtemperatures. The lengths of the products, in nucleotides, areindicated.

Surprisingly, cleavage by DNAPTaq requires neither a primer nor dNTPs(see FIG. 9A). Thus, the 5′ nuclease activity can be uncoupled frompolymerization. Nuclease activity requires magnesium ions, thoughmanganese ions can be substituted, albeit with potential changes inspecificity and activity. Neither zinc nor calcium ions support thecleavage reaction. The reaction occurs over a broad temperature range,from 25° C. to 85° C., with the rate of cleavage increasing at highertemperatures.

Still referring to FIG. 9, the primer is not elongated in the absence ofadded dNTPs. However, the primer influences both the site and the rateof cleavage of the hairpin. The change in the site of cleavage (FIG. 9A)apparently results from disruption of a short duplex formed between thearms of the DNA substrate. In the absence of primer, the sequencesindicated by underlining in FIG. 6 could pair, forming an extendedduplex. Cleavage at the end of the extended duplex would release the 11nucleotide fragment seen on the FIG. 9A lanes with no added primer.Addition of excess primer (FIG. 9A, lanes 3 and 4) or incubation at anelevated temperature (FIG. 9B) disrupts the short extension of theduplex and results in a longer 5′ arm and, hence, longer cleavageproducts.

The location of the 3′ end of the primer can influence the precise siteof cleavage. Electrophoretic analysis revealed that in the absence ofprimer (FIG. 9B), cleavage occurs at the end of the substrate duplex(either the extended or shortened form, depending on the temperature)between the first and second base pairs. When the primer extends up tothe base of the duplex, cleavage also occurs one nucleotide into theduplex. However, when a gap of four or six nucleotides exists betweenthe 3′ end of the primer and the substrate duplex, the cleavage site isshifted four to six nucleotides in the 5′ direction.

FIG. 10 describes the kinetics of cleavage in the presence (FIG. 10A) orabsence (FIG. 10B) of a primer oligonucleotide. The reactions were runat 55° C. with either 50 mM KCl (FIG. 10A) or 20 mM KCl (FIG. 10B). Thereaction products were resolved by denaturing polyacrylamide gelelectrophoresis and the lengths of the products, in nucleotides, areindicated. “M”, indicating a marker, is a 5′ end-labeled 19-ntoligonucleotide. Under these salt conditions, FIGS. 10A and 10B indicatethat the reaction appears to be about twenty times faster in thepresence of primer than in the absence of primer. This effect on theefficiency may be attributable to proper alignment and stabilization ofthe enzyme on the substrate.

The relative influence of primer on cleavage rates becomes much greaterwhen both reactions are run in 50 mM KCl. In the presence of primer, therate of cleavage increases with KCl concentration, up to about 50 mM.However, inhibition of this reaction in the presence of primer isapparent at 100 mM and is complete at 150 mM KCl. In contrast, in theabsence of primer the rate is enhanced by concentration of KCl up to 20mM, but it is reduced at concentrations above 30 mM. At 50 mM KCl, thereaction is almost completely inhibited. The inhibition of cleavage byKCl in the absence of primer is affected by temperature, being morepronounced at lower temperatures.

Recognition of the 5′ end of the arm to be cut appears to be animportant feature of substrate recognition. Substrates that lack a free5′ end, such as circular M13 DNA, cannot be cleaved under any conditionstested. Even with substrates having defined 5′ arms, the rate ofcleavage by DNAPTaq is influenced by the length of the arm. In thepresence of primer and 50 mM KCl, cleavage of a 5′ extension that is 27nucleotides long is essentially complete within 2 minutes at 55° C. Incontrast, cleavages of molecules with 5′ arms of 84 and 188 nucleotidesare only about 90% and 40% complete after 20 minutes. Incubation athigher temperatures reduces the inhibitory effects of long extensionsindicating that secondary structure in the 5′ arm or a heat-labilestructure in the enzyme may inhibit the reaction. A mixing experiment,run under conditions of substrate excess, shows that the molecules withlong arms do not preferentially tie up the available enzyme innon-productive complexes. These results may indicate that the 5′nuclease domain gains access to the cleavage site at the end of thebifurcated duplex by moving down the 5′ arm from one end to the other.Longer 5′ arms would be expected to have more adventitious secondarystructures (particularly when KCl concentrations are high), which wouldbe likely to impede this movement.

Cleavage does not appear to be inhibited by long 3′ arms of either thesubstrate strand target molecule or pilot nucleic acid, at least up to 2kilobases. At the other extreme, 3′ arms of the pilot nucleic acid asshort as one nucleotide can support cleavage in a primer-independentreaction, albeit inefficiently. Fully paired oligonucleotides do notelicit cleavage of DNA templates during primer extension.

The ability of DNAPTaq to cleave molecules even when the complementarystrand contains only one unpaired 3′ nucleotide may be useful inoptimizing allele-specific PCR. PCR primers that have unpaired 3′ endscould act as pilot oligonucleotides to direct selective cleavage ofunwanted templates during preincubation of potential template-primercomplexes with DNAPTaq in the absence of nucleoside triphosphates.

B. 5′ Nuclease Activities of Other DNAPs

To determine whether other 5′ nucleases in other DNAPs would be suitablefor the present invention, an array of enzymes, several of which werereported in the literature to be free of apparent 5′ nuclease activity,were examined. The ability of these other enzymes to cleave nucleicacids in a structure-specific manner was tested using the hairpinsubstrate shown in FIG. 6 under conditions reported to be optimal forsynthesis by each enzyme.

DNAPEc1 and DNAP Klenow were obtained from Promega Corporation; the DNAPof Pyrococcus furious (“Pfu”, Bargseid et al., Strategies 4:34 (1991))was from Stratagene; the DNAP of Thermococcus litoralis (“Tli”, VENT(exo-), Perler et al., Proc. Natl. Acad. Sci. USA 89:5577 (1992)) wasfrom New England Biolabs; the DNAP of Thermus flavus (“Tfl”, Kaledin etal., Biokhimiya 46:1576 (1981)) was from Epicentre Technologies; and theDNAP of Thermus thermophilus (“Tth”, Carballeira et al., Biotechniques9:276 (1990); Myers et al., Biochem. 30:7661 (1991)) was from U.S.Biochemicals.

0.5 units of each DNA polymerase was assayed in a 20 μl reaction, usingeither the buffers supplied by the manufacturers for theprimer-dependent reactions, or mM Tris.Cl, pH 8.5, 1.5 mM MgCl₂, and 20mM KCl. Reaction mixtures were at held 72° C. before the addition ofenzyme.

FIG. 11 is an autoradiogram recording the results of these tests. FIG.11A demonstrates reactions of endonucleases of DNAPs of severalthermophilic bacteria. The reactions were incubated at 55° C. for 10minutes in the presence of primer or at 72° C. for 30 minutes in theabsence of primer, and the products were resolved by denaturingpolyacrylamide gel electrophoresis. The lengths of the products, innucleotides, are indicated. FIG. 11B demonstrates endonucleolyticcleavage by the 5′ nuclease of DNAPEc1. The DNAPEc1 and DNAP Klenowreactions were incubated for 5 minutes at 37° C. Note the light band ofcleavage products of 25 and 11 nucleotides in the DNAPEc1 lanes (made inthe presence and absence of primer, respectively). FIG. 7B alsodemonstrates DNAPTaq reactions in the presence (+) or absence (−) ofprimer. These reactions were run in 50 mM and 20 mM KCl, respectively,and were incubated at 55° C. for 10 minutes.

Referring to FIG. 11A, DNAPs from the eubacteria Thermus thermophilusand Thermus flavus cleave the substrate at the same place as DNAPTaq,both in the presence and absence of primer. In contrast, DNAPs from thearchaebacteria Pyrococcus furiosus and Thermococcus litoralis are unableto cleave the substrates endonucleolytically. The DNAPs from Pyrococcusfurious and Thermococcus litoralis share little sequence homology witheubacterial enzymes (Ito et al., Nucl. Acids Res. 19:4045 (1991); Mathuret al., Nucl. Acids. Res. 19:6952 (1991); see also Perler et al.).Referring to FIG. 11B, DNAPEc1 also cleaves the substrate, but theresulting cleavage products are difficult to detect unless the 3′exonuclease is inhibited. The amino acid sequences of the 5′ nucleasedomains of DNAPEc1 and DNAPTaq are about 38% homologous (Gelfand,supra).

The 5′ nuclease domain of DNAPTaq also shares about 19% homology withthe 5′ exonuclease encoded by gene 6 of bacteriophage T7 (Dunn et al.,J. Mol. Biol. 166:477 (1983)). This nuclease, which is not covalentlyattached to a DNAP polymerization domain, is also able to cleave DNAendonucleolytically, at a site similar or identical to the site that iscut by the 5′ nucleases described above, in the absence of addedprimers.

C. Transcleavage

The ability of a 5′ nuclease to be directed to cleave efficiently at anyspecific sequence was demonstrated in the following experiment. Apartially complementary oligonucleotide termed a “pilot oligonucleotide”was hybridized to sequences at the desired point of cleavage. Thenon-complementary part of the pilot oligonucleotide provided a structureanalogous to the 3′ arm of the template (see FIG. 6), whereas the 5′region of the substrate strand became the 5′ arm. A primer was providedby designing the 3′ region of the pilot so that it would fold on itselfcreating a short hairpin with a stabilizing tetra-loop (Antao et al.,Nucl. Acids Res. 19:5901 (1991)). Two pilot oligonucleotides are shownin FIG. 12A. Oligonucleotides 19-12 (SEQ ID NO:18) and 30-12 (SEQ IDNO:19) are 31 or 42 or nucleotides long, respectively. However,oligonucleotides 19-12 (SEQ ID NO:18) and 34-19 (SEQ ID NO:19) have only19 and 30 nucleotides, respectively, that are complementary to differentsequences in the substrate strand. The pilot oligonucleotides arecalculated to melt off their complements at about 50° C. (19-12) andabout 75° C. (30-12). Both pilots have 12 nucleotides at their 3′ ends,which act as 3′ arms with base-paired primers attached.

To demonstrate that cleavage could be directed by a pilotoligonucleotide, we incubated a single-stranded target DNA with DNAPTaqin the presence of two potential pilot oligonucleotides. Thetranscleavage reactions, where the target and pilot nucleic acids arenot covalently linked, includes 0.01 pmoles of single end-labeledsubstrate DNA, 1 unit of DNAPTaq and 5 pmoles of pilot oligonucleotidein a volume of 20 μl of the same buffers. These components were combinedduring a one minute incubation at 95° C., to denature the PCR-generateddouble-stranded substrate DNA, and the temperatures of the reactionswere then reduced to their final incubation temperatures.Oligonucleotides 30-12 and 19-12 can hybridize to regions of thesubstrate DNAs that are 85 and 27 nucleotides from the 5′ end of thetargeted strand.

FIG. 21 shows the complete 206-mer sequence (SEQ ID NO:32). The 206-merwas generated by PCR. The M13/pUC 24-mer reverse sequencing (−48) primerand the M13/pUC sequencing (−47) primer from New England Biolabs(catalogue nos. 1233 and 1224 respectively) were used (50 pmoles each)with the pGEM3z(f+) plasmid vector (Promega Corp.) as template (10 ng)containing the target sequences. The conditions for PCR were as follows:50 μM of each dNTP and 2.5 units of Taq DNA polymerase in 100 μl of 20mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl with 0.05% TWEEN-20 and0.05% NP-40. Reactions were cycled 35 times through 95° C. for 45seconds, 63° C. for 45 seconds, then 72° C. for 75 seconds. Aftercycling, reactions were finished off with an incubation at 72° C. for 5minutes. The resulting fragment was purified by electrophoresis througha 6% polyacrylamide gel (29:1 cross link) in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA, visualized by ethidium bromidestaining or autoradiography, excised from the gel, eluted by passivediffusion, and concentrated by ethanol precipitation.

Cleavage of the substrate DNA occurred in the presence of the pilotoligonucleotide 19-12 at 50° C. (FIG. 12B, lanes 1 and 7) but not at 75°C. (lanes 4 and 10). In the presence of oligonucleotide 30-12 cleavagewas observed at both temperatures. Cleavage did not occur in the absenceof added oligonucleotides (lanes 3, 6 and 12) or at about 80° C. eventhough at 50° C. adventitious structures in the substrate allowedprimer-independent cleavage in the absence of KCl (FIG. 12B, lane 9). Anon-specific oligonucleotide with no complementarity to the substrateDNA did not direct cleavage at 50° C., either in the absence or presenceof 50 mM KCl (lanes 13 and 14). Thus, the specificity of the cleavagereactions can be controlled by the extent of complementarity to thesubstrate and by the conditions of incubation.

D. Cleavage Of RNA

An shortened RNA version of the sequence used in the transcleavageexperiments discussed above was tested for its ability to serve as asubstrate in the reaction. The RNA is cleaved at the expected place, ina reaction that is dependent upon the presence of the pilotoligonucleotide. The RNA substrate, made by T7 RNA polymerase in thepresence of (α-³²P)UTP, corresponds to a truncated version of the DNAsubstrate used in FIG. 12B. Reaction conditions were similar to those inused for the DNA substrates described above, with 50 mM KCl; incubationwas for 40 minutes at 55° C. The pilot oligonucleotide used is termed30-0 (SEQ ID NO:20) and is shown in FIG. 13A.

The results of the cleavage reaction is shown in FIG. 13B. The reactionwas run either in the presence or absence of DNAPTaq or pilotoligonucleotide as indicated in FIG. 13B.

Strikingly, in the case of RNA cleavage, a 3′ arm is not required forthe pilot oligonucleotide. It is very unlikely that this cleavage is dueto previously described RNaseH, which would be expected to cut the RNAin several places along the 30 base-pair long RNA-DNA duplex. The 5′nuclease of DNAPTaq is a structure-specific RNaseH that cleaves the RNAat a single site near the 5′ end of the heteroduplexed region.

It is surprising that an oligonucleotide lacking a 3′ arm is able to actas a pilot in directing efficient cleavage of an RNA target because sucholigonucleotides are unable to direct efficient cleavage of DNA targetsusing native DNAPs. However, some 5′ nucleases of the present invention(for example, clones E, F and G of FIG. 4) can cleave DNA in the absenceof a 3′ arm. In other words, a non-extendable cleavage structure is notrequired for specific cleavage with some 5′ nucleases of the presentinvention derived from thermostable DNA polymerases.

We tested whether cleavage of an RNA template by DNAPTaq in the presenceof a fully complementary primer could help explain why DNAPTaq is unableto extend a DNA oligonucleotide on an RNA template, in a reactionresembling that of reverse transcriptase. Another thermophilic DNAP,DNAPTth, is able to use RNA as a template, but only in the presence ofMn++, so we predicted that this enzyme would not cleave RNA in thepresence of this cation. Accordingly, we incubated an RNA molecule withan appropriate pilot oligonucleotide in the presence of DNAPTaq orDNAPTth, in buffer containing either Mg++ or Mn++. As expected, bothenzymes cleaved the RNA in the presence of Mg++. However, DNAPTaq, butnot DNAPTth, degraded the RNA in the presence of Mn++. We conclude thatthe 5′ nuclease activities of many DNAPs may contribute to theirinability to use RNA as templates.

Example 2 Generation of 5′ Nucleases from Thermostable DNA Polymerases

Thermostable DNA polymerases were generated which have reduced syntheticactivity, an activity that is an undesirable side-reaction during DNAcleavage in the detection assay of the invention, yet have maintainedthermostable nuclease activity. The result is a thermostable polymerasewhich cleaves nucleic acids DNA with extreme specificity.

Type A DNA polymerases from eubacteria of the genus Thermus shareextensive protein sequence identity (90% in the polymerization domain,using the Lipman-Pearson method in the DNA analysis software fromDNAStar, WI) and behave similarly in both polymerization and nucleaseassays. Therefore, we have used the genes for the DNA polymerase ofThermus aquaticus (DNAPTaq) and Thermus flavus (DNAPTfl) asrepresentatives of this class. Polymerase genes from other eubacterialorganisms, such as Thermus thermophilus, Thermus sp., Thermotogamaritima, Thermosipho africanus and Bacillus stearothermophilus areequally suitable. The DNA polymerases from these thermophilic organismsare capable of surviving and performing at elevated temperatures, andcan thus be used in reactions in which temperature is used as aselection against non-specific hybridization of nucleic acid strands.

The restriction sites used for deletion mutagenesis, described below,were chosen for convenience. Different sites situated with similarconvenience are available in the Thermus thermophilus gene and can beused to make similar constructs with other Type A polymerase genes fromrelated organisms.

A. Creation of 5′ Nuclease Constructs

1. Modified DNAPTaq Genes

The first step was to place a modified gene for the Taq DNA polymeraseon a plasmid under control of an inducible promoter. The modified Taqpolymerase gene was isolated as follows: The Taq DNA polymerase gene wasamplified by polymerase chain reaction from genomic DNA from Thermusaquaticus, strain YT-1 (Lawyer et al., supra), using as primers theoligonucleotides described in SEQ ID NOS:13-14. The resulting fragmentof DNA has a recognition sequence for the restriction endonuclease EcoRIat the 5′ end of the coding sequence and a BglII sequence at the 3′ end.Cleavage with BglII leaves a 5′ overhang or “sticky end” that iscompatible with the end generated by BamHI. The PCR-amplified DNA wasdigested with EcoRI and BamHI. The 2512 by fragment containing thecoding region for the polymerase gene was gel purified and then ligatedinto a plasmid which contains an inducible promoter.

In one embodiment of the invention, the pTTQ18 vector, which containsthe hybrid trp-lac (tac) promoter, was used (M. J. R. Stark, Gene 5:255(1987)) and shown in FIG. 14. The tac promoter is under the control ofthe E. coli lac repressor. Repression allows the synthesis of the geneproduct to be suppressed until the desired level of bacterial growth hasbeen achieved, at which point repression is removed by addition of aspecific inducer, isopropyl-β-D-thiogalactopyranoside (IPTG). Such asystem allows the expression of foreign proteins that may slow orprevent growth of transformants.

Bacterial promoters, such as tac, may not be adequately suppressed whenthey are present on a multiple copy plasmid. If a highly toxic proteinis placed under control of such a promoter, the small amount ofexpression leaking through can be harmful to the bacteria. In anotherembodiment of the invention, another option for repressing synthesis ofa cloned gene product was used. The non-bacterial promoter, frombacteriophage T7, found in the plasmid vector series pET-3 was used toexpress the cloned mutant Taq polymerase genes (FIG. 15; Studier andMoffatt, J. Mol. Biol. 189:113 (1986)). This promoter initiatestranscription only by T7 RNA polymerase. In a suitable strain, such asBL21(DE3)pLYS, the gene for this RNA polymerase is carried on thebacterial genome under control of the lac operator. This arrangement hasthe advantage that expression of the multiple copy gene (on the plasmid)is completely dependent on the expression of T7 RNA polymerase, which iseasily suppressed because it is present in a single copy.

For ligation into the pTTQ18 vector (FIG. 14), the PCR product DNAcontaining the Taq polymerase coding region (mutTaq, clone 4B, SEQ IDNO:21) was digested with EcoRI and BglII and this fragment was ligatedunder standard “sticky end” conditions (Sambrook et al. MolecularCloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp.1.63-1.69 (1989)) into the EcoRI and BamHI sites of the plasmid vectorpTTQ18. Expression of this construct yields a translational fusionproduct in which the first two residues of the native protein (Met-Arg)are replaced by three from the vector (Met-Asn-Ser), but the remainderof the natural protein would not change. The construct was transformedinto the JM109 strain of E. coli and the transformants were plated underincompletely repressing conditions that do not permit growth of bacteriaexpressing the native protein. These plating conditions allow theisolation of genes containing pre-existing mutations, such as those thatresult from the infidelity of Taq polymerase during the amplificationprocess.

Using this amplification/selection protocol, we isolated a clone(depicted in FIG. 4B) containing a mutated Taq polymerase gene (mutTaq,clone 4B). The mutant was first detected by its phenotype, in whichtemperature-stable 5′ nuclease activity in a crude cell extract wasnormal, but polymerization activity was almost absent (approximatelyless than 1% of wild type Taq polymerase activity).

DNA sequence analysis of the recombinant gene showed that it had changesin the polymerase domain resulting in two amino acid substitutions: an Ato G change at nucleotide position 1394 causes a Glu to Gly change atamino acid position 465 (numbered according to the natural nucleic andamino acid sequences, SEQ ID NOS:1 and 4) and another A to G change atnucleotide position 2260 causes a Gln to Arg change at amino acidposition 754. Because the Gln to Gly mutation is at a nonconservedposition and because the Glu to Arg mutation alters an amino acid thatis conserved in virtually all of the known Type A polymerases, thislatter mutation is most likely the one responsible for curtailing thesynthesis activity of this protein. The nucleotide sequence for the FIG.4B construct is given in SEQ ID NO:21. The corresponding amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:21 is listed inSEQ ID NO:85.

Subsequent derivatives of DNAPTaq constructs were made from the mutTaqgene, thus, they all bear these amino acid substitutions in addition totheir other alterations, unless these particular regions were deleted.These mutated sites are indicated by black boxes at these locations inthe diagrams in FIG. 4. In FIG. 4, the designation “3′ Exo” is used toindicate the location of the 3′ exonuclease activity associated withType A polymerases which is not present in DNAPTaq. All constructsexcept the genes shown in FIGS. 4E, F and G were made in the pTTQ18vector.

The cloning vector used for the genes in FIGS. 4E and F was from thecommercially available pET-3 series, described above. Though this vectorseries has only a BamHI site for cloning downstream of the T7 promoter,the series contains variants that allow cloning into any of the threereading frames. For cloning of the PCR product described above, thevariant called pET-3c was used (FIG. 15). The vector was digested withBamHI, dephosphorylated with calf intestinal phosphatase, and the stickyends were filled in using the Klenow fragment of DNAPEc1 and dNTPs. Thegene for the mutant Taq DNAP shown in FIG. 4B (mutTaq, clone 4B) wasreleased from pTTQ18 by digestion with EcoRI and SalI, and the “stickyends” were filled in as was done with the vector. The fragment wasligated to the vector under standard blunt-end conditions (Sambrook etal., Molecular Cloning, supra), the construct was transformed into theBL21(DE3)pLYS strain of E. coli, and isolates were screened to identifythose that were ligated with the gene in the proper orientation relativeto the promoter. This construction yields another translational fusionproduct, in which the first two amino acids of DNAPTaq (Met-Arg) arereplaced by 13 from the vector plus two from the PCR primer(Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Ile-Asn-Ser) (SEQ IDNO:29).

Our goal was to generate enzymes that lacked the ability to synthesizeDNA, but retained the ability to cleave nucleic acids with a 5′ nucleaseactivity. The act of primed, templated synthesis of DNA is actually acoordinated series of events, so it is possible to disable DNA synthesisby disrupting one event while not affecting the others. These stepsinclude, but are not limited to, primer recognition and binding, dNTPbinding and catalysis of the inter-nucleotide phosphodiester bond. Someof the amino acids in the polymerization domain of DNAPEcI have beenlinked to these functions, but the precise mechanisms are as yet poorlydefined.

One way of destroying the polymerizing ability of a DNA polymerase is todelete all or part of the gene segment that encodes that domain for theprotein, or to otherwise render the gene incapable of making a completepolymerization domain. Individual mutant enzymes may differ from eachother in stability and solubility both inside and outside cells. Forinstance, in contrast to the 5′ nuclease domain of DNAPEcI, which can bereleased in an active form from the polymerization domain by gentleproteolysis (Setlow and Kornberg, J. Biol. Chem. 247:232 (1972)), theThermus nuclease domain, when treated similarly, becomes less solubleand the cleavage activity is often lost.

Using the mutant gene shown in FIG. 4B as starting material, severaldeletion constructs were created. All cloning technologies were standard(Sambrook et al., supra) and are summarized briefly, as follows:

FIG. 4C: The mutTaq construct was digested with PstI, which cuts oncewithin the polymerase coding region, as indicated, and cuts immediatelydownstream of the gene in the multiple cloning site of the vector. Afterrelease of the fragment between these two sites, the vector wasre-ligated, creating an 894-nucleotide deletion, and bringing into framea stop codon 40 nucleotides downstream of the junction. The nucleotidesequence of this 5′ nuclease (clone 4C) is given in SEQ ID NO:9. Thecorresponding amino acid sequence encoded by the nucleotide sequence ofSEQ ID NO:9 is listed in SEQ ID NO:86.

FIG. 4D: The mutTaq construct was digested with NheI, which cuts once inthe gene at position 2047. The resulting four-nucleotide 5′ overhangingends were filled in, as described above, and the blunt ends werere-ligated. The resulting four-nucleotide insertion changes the readingframe and causes termination of translation ten amino acids downstreamof the mutation. The nucleotide sequence of this 5′ nuclease (clone 4D)is given in SEQ ID NO:10. The corresponding amino acid sequence encodedby the nucleotide sequence of SEQ ID NO:10 is listed in SEQ ID NO:87.

FIG. 4E: The entire mutTaq gene was cut from pTTQ18 using EcoRI and SalIand cloned into pET-3c, as described above. This clone was digested withBstXI and XcmI, at unique sites that are situated as shown in FIG. 4E.The DNA was treated with the Klenow fragment of DNAPEc1 and dNTPs, whichresulted in the 3′ overhangs of both sites being trimmed to blunt ends.These blunt ends were ligated together, resulting in an out-of-framedeletion of 1540 nucleotides. An in-frame termination codon occurs 18triplets past the junction site. The nucleotide sequence of this 5′nuclease (clone 4E) is given in SEQ ID NO:11 (The corresponding aminoacid sequence encoded by the nucleotide sequence of SEQ ID NO:11 islisted in SEQ ID NO:88)., with the appropriate leader sequence given inSEQ ID NO:30 (The corresponding amino acid sequence encoded by thenucleotide sequence of SEQ ID NO:30 is listed in SEQ ID NO:89). It isalso referred to as the enzyme CLEAVASE BX.

FIG. 4F: The entire mutTaq gene was cut from pTTQ18 using EcoRI and SalIand cloned into pET-3c, as described above. This clone was digested withBstXI and BamHI, at unique sites that are situated as shown in thediagram. The DNA was treated with the Klenow fragment of DNAPEc1 anddNTPs, which resulted in the 3′ overhang of the BstX I site beingtrimmed to a blunt end, while the 5′ overhang of the Bam HI site wasfilled in to make a blunt end. These ends were ligated together,resulting in an in-frame deletion of 903 nucleotides. The nucleotidesequence of the 5′ nuclease (clone 4F) is given in SEQ ID NO:12. It isalso referred to as the enzyme CLEAVASE BB. The corresponding amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:12 is listed inSEQ ID NO:90.

FIG. 4G: This polymerase is a variant of that shown in FIG. 4E. It wascloned in the plasmid vector pET-21 (Novagen). The non-bacterialpromoter from bacteriophage T7, found in this vector, initiatestranscription only by T7 RNA polymerase. See Studier and Moffatt, supra.In a suitable strain, such as (DES)pLYS, the gene for this RNApolymerase is carried on the bacterial genome under control of the lacoperator. This arrangement has the advantage that expression of themultiple copy gene (on the plasmid) is completely dependent on theexpression of T7 RNA polymerase, which is easily suppressed because itis present in a single copy. Because the expression of these mutantgenes is under this tightly controlled promoter, potential problems oftoxicity of the expressed proteins to the host cells are less of aconcern.

The pET-21 vector also features a “His-Tag”, a stretch of sixconsecutive histidine residues that are added on the carboxy terminus ofthe expressed proteins. The resulting proteins can then be purified in asingle step by metal chelation chromatography, using a commerciallyavailable (Novagen) column resin with immobilized Ni⁺⁺ ions. The 2.5 mlcolumns are reusable, and can bind up to 20 mg of the target proteinunder native or denaturing (guanidine-HCl or urea) conditions.

E. coli (DES)pLYS cells are transformed with the constructs describedabove using standard transformation techniques, and used to inoculate astandard growth medium (e.g., Luria-Bertani broth). Production of T7 RNApolymerase is induced during log phase growth by addition of IPTG andincubated for a further 12 to 17 hours. Aliquots of culture are removedboth before and after induction and the proteins are examined bySDS-PAGE. Staining with Coomassie Blue allows visualization of theforeign proteins if they account for about 3-5% of the cellular proteinand do not co-migrate with any of the major host protein bands. Proteinsthat co-migrate with major host proteins must be expressed as more than10% of the total protein to be seen at this stage of analysis.

Some mutant proteins are sequestered by the cells into inclusion bodies.These are granules that form in the cytoplasm when bacteria are made toexpress high levels of a foreign protein, and they can be purified froma crude lysate, and analyzed by SDS-PAGE to determine their proteincontent. If the cloned protein is found in the inclusion bodies, it mustbe released to assay the cleavage and polymerase activities. Differentmethods of solubilization may be appropriate for different proteins, anda variety of methods are known. See e.g., Builder & Ogez, U.S. Pat. No.4,511,502 (1985); Olson, U.S. Pat. No. 4,518,526 (1985); Olson & Pai,U.S. Pat. No. 4,511,503 (1985); Jones et al., U.S. Pat. No. 4,512,922(1985), all of which are hereby incorporated by reference.

The solubilized protein is then purified on the Ni⁺⁺ column as describedabove, following the manufacturers instructions (Novagen). The washedproteins are eluted from the column by a combination of imidazolecompetitor (1 M) and high salt (0.5 M NaCl), and dialyzed to exchangethe buffer and to allow denatured proteins to refold. Typical recoveriesresult in approximately 20 μg of specific protein per ml of startingculture. The DNAP mutant is referred to as the enzyme CLEAVASE BN andthe sequence is given in SEQ ID NO:31. The corresponding amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:31 is listed inSEQ ID NO:91.

2. Modified DNAPTfl Gene

The DNA polymerase gene of Thermus flavus was isolated from the “T.flavus” AT-62 strain obtained from the American Type Tissue Collection(ATCC 33923). This strain has a different restriction map then does theT. flavus strain used to generate the sequence published by Akhmetzjanovand Vakhitov, supra. The published sequence is listed as SEQ ID NO:2. Nosequence data has been published for the DNA polymerase gene from theAT-62 strain of T. flavus.

Genomic DNA from T. flavus was amplified using the same primers used toamplify the T. aquaticus DNA polymerase gene (SEQ ID NOS:13-14). Theapproximately 2500 base pair PCR fragment was digested with EcoRI andBamHI. The over-hanging ends were made blunt with the Klenow fragment ofDNAPEc1 and dNTPs. The resulting approximately 1800 base pair fragmentcontaining the coding region for the N-terminus was ligated into pET-3c,as described above. This construct, clone 5B, is depicted in FIG. 5B.The wild type T. flavus DNA polymerase gene is depicted in FIG. 5A. InFIG. 5, the designation “3′ Exo” is used to indicate the location of the3′ exonuclease activity associated with Type A polymerases which is notpresent in DNAPTf1. The 5B clone has the same leader amino acids as dothe DNAPTaq clones 4E and F which were cloned into pET-3c; it is notknown precisely where translation termination occurs, but the vector hasa strong transcription termination signal immediately downstream of thecloning site.

B. Growth and Induction of Transformed Cells

Bacterial cells were transformed with the constructs described aboveusing standard transformation techniques and used to inoculate 2 mls ofa standard growth medium (e.g., Luria-Bertani broth). The resultingcultures were incubated as appropriate for the particular strain used,and induced if required for a particular expression system. For all ofthe constructs depicted in FIGS. 4 and 5, the cultures were grown to anoptical density (at 600 nm wavelength) of 0.5 OD.

To induce expression of the cloned genes, the cultures were brought to afinal concentration of 0.4 mM IPTG and the incubations were continuedfor 12 to 17 hours. 50 μl aliquots of each culture were removed bothbefore and after induction and were combined with 20 μl of a standardgel loading buffer for sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). Subsequent staining with Coomassie Blue(Sambrook et al., supra) allows visualization of the foreign proteins ifthey account for about 3-5% of the cellular protein and do notco-migrate with any of the major E. coli protein bands. Proteins that doco-migrate with a major host protein must be expressed as more than 10%of the total protein to be seen at this stage of analysis.

C. Heat Lysis And Fractionation

Expressed thermostable proteins, i.e., the 5′ nucleases, were isolatedby heating crude bacterial cell extracts to cause denaturation andprecipitation of the less stable E. coli proteins. The precipitated E.coli proteins were then, along with other cell debris, removed bycentrifugation. 1.7 mls of the culture were pelleted bymicrocentrifugation at 12,000 to 14,000 rpm for 30 to 60 seconds. Afterremoval of the supernatant, the cells were resuspended in 400 μl ofbuffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1 mM EDTA),re-centrifuged, then resuspended in 80 μl of buffer A with 4 mg/mllysozyme. The cells were incubated at room temperature for 15 minutes,then combined with 80 μl of buffer B (10 mM Tris-HCl, pH 7.9, 50 mM KCl,1 mM EDTA, 1 mM PMSF, 0.5% TWEEN-20, 0.5% NONIDET-P40).

This mixture was incubated at 75° C. for 1 hour to denature andprecipitate the host proteins. This cell extract was centrifuged at14,000 rpm for 15 minutes at 4° C., and the supernatant was transferredto a fresh tube. An aliquot of 0.5 to 1 μA of this supernatant was useddirectly in each test reaction, and the protein content of the extractwas determined by subjecting 7 μA to electrophoretic analysis, as above.The native recombinant Taq DNA polymerase (Englke, Anal. Biochem 191:396(1990)), and the double point mutation protein shown in FIG. 4B are bothsoluble and active at this point.

The foreign protein may not be detected after the heat treatments due tosequestration of the foreign protein by the cells into inclusion bodies.These are granules that form in the cytoplasm when bacteria are made toexpress high levels of a foreign protein, and they can be purified froma crude lysate, and analyzed SDS PAGE to determine their proteincontent. Many methods have been described in the literature, and oneapproach is described below.

D. Isolation and Solubilization of Inclusion Bodies

A small culture was grown and induced as described above. A 1.7 mlaliquot was pelleted by brief centrifugation, and the bacterial cellswere resuspended in 100 μl of Lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mMEDTA, 100 mM NaCl). 2.5 μl of 20 mM PMSF were added for a finalconcentration of 0.5 mM, and lysozyme was added to a concentration of1.0 mg/ml. The cells were incubated at room temperature for 20 minutes,deoxycholic acid was added to 1 mg/ml (1 μl of 100 mg/ml solution), andthe mixture was further incubated at 37° C. for about 15 minutes oruntil viscous. DNAseI was added to 10 μg/ml and the mixture wasincubated at room temperature for about 30 minutes or until it was nolonger viscous.

From this mixture the inclusion bodies were collected by centrifugationat 14,000 rpm for 15 minutes at 4° C., and the supernatant wasdiscarded. The pellet was resuspended in 100 μl of lysis buffer with 10mM EDTA (pH 8.0) and 0.5% TRITON X-100. After 5 minutes at roomtemperature, the inclusion bodies were pelleted as before, and thesupernatant was saved for later analysis. The inclusion bodies wereresuspended in 50 μl of distilled water, and 5 μl was combined with SDSgel loading buffer (which dissolves the inclusion bodies) and analyzedelectrophoretically, along with an aliquot of the supernatant.

If the cloned protein is found in the inclusion bodies, it may bereleased to assay the cleavage and polymerase activities and the methodof solubilization must be compatible with the particular activity.Different methods of solubilization may be appropriate for differentproteins, and a variety of methods are discussed in Molecular Cloning(Sambrook et al., supra). The following is an adaptation we have usedfor several of our isolates.

20 μl of the inclusion body-water suspension were pelleted bycentrifugation at 14,000 rpm for 4 minutes at room temperature, and thesupernatant was discarded. To further wash the inclusion bodies, thepellet was resuspended in 20 μl of lysis buffer with 2M urea, andincubated at room temperature for one hour. The washed inclusion bodieswere then resuspended in 2 μl of lysis buffer with 8M urea; the solutionclarified visibly as the inclusion bodies dissolved. Undissolved debriswas removed by centrifugation at 14,000 rpm for 4 minutes at roomtemperature, and the extract supernatant was transferred to a freshtube.

To reduce the urea concentration, the extract was diluted into KH₂PO₄. Afresh tube was prepared containing 180 μl of 50 mM KH₂PO₄, pH 9.5, 1 mMEDTA and 50 mM NaCl. A 2 μl aliquot of the extract was added andvortexed briefly to mix. This step was repeated until all of the extracthad been added for a total of 10 additions. The mixture was allowed tosit at room temperature for 15 minutes, during which time someprecipitate often forms. Precipitates were removed by centrifugation at14,000 rpm, for 15 minutes at room temperature, and the supernatant wastransferred to a fresh tube. To the 200 μl of protein in the KH₂PO₄solution, 140-200 μl of saturated (NH₄)₂SO₄ were added, so that theresulting mixture was about 41% to 50% saturated (NH₄)₂SO₄. The mixturewas chilled on ice for 30 minutes to allow the protein to precipitate,and the protein was then collected by centrifugation at 14,000 rpm, for4 minutes at room temperature. The supernatant was discarded, and thepellet was dissolved in 20 μl Buffer C (20 mM HEPES, pH 7.9, 1 mM EDTA,0.5% PMSF, 25 mM KCl and 0.5% each of TWEEN-20 and NONIDET P 40). Theprotein solution was centrifuged again for 4 minutes to pellet insolublematerials, and the supernatant was removed to a fresh tube. The proteincontents of extracts prepared in this manner were visualized byresolving 1-4 μl by SDS-PAGE; 0.5 to 1 μl of extract was tested in thecleavage and polymerization assays as described.

E. Protein Analysis for Presence of Nuclease and Synthetic Activity

The 5′ nucleases described above and shown in FIGS. 4 and 5 wereanalyzed by the following methods.

1. Structure Specific Nuclease Assay

A candidate modified polymerase is tested for 5′ nuclease activity byexamining its ability to catalyze structure-specific cleavages. By theterm “cleavage structure” as used herein, is meant a nucleic acidstructure which is a substrate for cleavage by the 5′ nuclease activityof a DNAP.

The polymerase is exposed to test complexes that have the structuresshown in FIG. 16. Testing for 5′ nuclease activity involves threereactions: 1) a primer-directed cleavage (FIG. 16B) is performed becauseit is relatively insensitive to variations in the salt concentration ofthe reaction and can, therefore, be performed in whatever soluteconditions the modified enzyme requires for activity; this is generallythe same conditions preferred by unmodified polymerases; 2) a similarprimer-directed cleavage is performed in a buffer which permitsprimer-independent cleavage, i.e., a low salt buffer, to demonstratethat the enzyme is viable under these conditions; and 3) aprimer-independent cleavage (FIG. 16A) is performed in the same low saltbuffer.

The bifurcated duplex is formed between a substrate strand and atemplate strand as shown in FIG. 16. By the term “substrate strand” asused herein, is meant that strand of nucleic acid in which the cleavagemediated by the 5′ nuclease activity occurs. The substrate strand isalways depicted as the top strand in the bifurcated complex which servesas a substrate for 5′ nuclease cleavage (FIG. 16). By the term “templatestrand” as used herein, is meant the strand of nucleic acid which is atleast partially complementary to the substrate strand and which annealsto the substrate strand to form the cleavage structure. The templatestrand is always depicted as the bottom strand of the bifurcatedcleavage structure (FIG. 16). If a primer (a short oligonucleotide of 19to 30 nucleotides in length) is added to the complex, as whenprimer-dependent cleavage is to be tested, it is designed to anneal tothe 3′ arm of the template strand (FIG. 16B). Such a primer would beextended along the template strand if the polymerase used in thereaction has synthetic activity.

The cleavage structure may be made as a single hairpin molecule, withthe 3′ end of the target and the 5′ end of the pilot joined as a loop asshown in FIG. 16E. A primer oligonucleotide complementary to the 3′ armis also required for these tests so that the enzyme's sensitivity to thepresence of a primer may be tested.

Nucleic acids to be used to form test cleavage structures can bechemically synthesized, or can be generated by standard recombinant DNAtechniques. By the latter method, the hairpin portion of the moleculecan be created by inserting into a cloning vector duplicate copies of ashort DNA segment, adjacent to each other but in opposing orientation.The double-stranded fragment encompassing this inverted repeat, andincluding enough flanking sequence to give short (about 20 nucleotides)unpaired 5′ and 3′ arms, can then be released from the vector byrestriction enzyme digestion, or by PCR performed with an enzyme lackinga 5′ exonuclease (e.g., the Stoffel fragment of AMPLITAQ DNA polymerase,VENT DNA polymerase).

The test DNA can be labeled on either end, or internally, with either aradioisotope, or with a non-isotopic tag. Whether the hairpin DNA is asynthetic single strand or a cloned double strand, the DNA is heatedprior to use to melt all duplexes. When cooled on ice, the structuredepicted in FIG. 16E is formed, and is stable for sufficient time toperform these assays.

To test for primer-directed cleavage (Reaction 1), a detectable quantityof the test molecule (typically 1-100 fmol of ³²P-labeled hairpinmolecule) and a 10 to 100-fold molar excess of primer are placed in abuffer known to be compatible with the test enzyme. For Reaction 2,where primer-directed cleavage is performed under condition which allowprimer-independent cleavage, the same quantities of molecules are placedin a solution that is the same as the buffer used in Reaction 1regarding pH, enzyme stabilizers (e.g., bovine serum albumin, nonionicdetergents, gelatin) and reducing agents (e.g., dithiothreitol,2-mercaptoethanol) but that replaces any monovalent cation salt with 20mM KCl; 20 mM KCl is the demonstrated optimum for primer-independentcleavage. Buffers for enzymes, such as DNAPEc1, that usually operate inthe absence of salt are not supplemented to achieve this concentration.To test for primer-independent cleavage (Reaction 3) the same quantityof the test molecule, but no primer, are combined under the same bufferconditions used for Reaction 2.

All three test reactions are then exposed to enough of the enzyme thatthe molar ratio of enzyme to test complex is approximately 1:1. Thereactions are incubated at a range of temperatures up to, but notexceeding, the temperature allowed by either the enzyme stability or thecomplex stability, whichever is lower, up to 80° C. for enzymes fromthermophiles, for a time sufficient to allow cleavage (10 to 60minutes). The products of Reactions 1, 2 and 3 are resolved bydenaturing polyacrylamide gel electrophoresis, and visualized byautoradiography or by a comparable method appropriate to the labelingsystem used. Additional labeling systems include chemiluminescencedetection, silver or other stains, blotting and probing and the like.The presence of cleavage products is indicated by the presence ofmolecules which migrate at a lower molecular weight than does theuncleaved test structure. These cleavage products indicate that thecandidate polymerase has structure-specific 5′ nuclease activity.

To determine whether a modified DNA polymerase has substantially thesame 5′ nuclease activity as that of the native DNA polymerase, theresults of the above-described tests are compared with the resultsobtained from these tests performed with the native DNA polymerase. By“substantially the same 5′ nuclease activity” we mean that the modifiedpolymerase and the native polymerase will both cleave test molecules inthe same manner. It is not necessary that the modified polymerase cleaveat the same rate as the native DNA polymerase.

Some enzymes or enzyme preparations may have other associated orcontaminating activities that may be functional under the cleavageconditions described above and that may interfere with 5′ nucleasedetection. Reaction conditions can be modified in consideration of theseother activities, to avoid destruction of the substrate, or othermasking of the 5′ nuclease cleavage and its products. For example, theDNA polymerase I of E. coli (Pol I), in addition to its polymerase and5′ nuclease activities, has a 3′ exonuclease that can degrade DNA in a3′ to 5′ direction. Consequently, when the molecule in FIG. 16E isexposed to this polymerase under the conditions described above, the 3′exonuclease quickly removes the unpaired 3′ arm, destroying thebifurcated structure required of a substrate for the 5′ exonucleasecleavage and no cleavage is detected. The true ability of Pol Ito cleavethe structure can be revealed if the 3′ exonuclease is inhibited by achange of conditions (e.g., pH), mutation, or by addition of acompetitor for the activity. Addition of 500 pmoles of a single-strandedcompetitor oligonucleotide, unrelated to the FIG. 16E structure, to thecleavage reaction with Pol I effectively inhibits the digestion of the3′ arm of the FIG. 16E structure without interfering with the 5′exonuclease release of the 5′ arm. The concentration of the competitoris not critical, but should be high enough to occupy the 3′ exonucleasefor the duration of the reaction.

Similar destruction of the test molecule may be caused by contaminantsin the candidate polymerase preparation. Several sets of the structurespecific nuclease reactions may be performed to determine the purity ofthe candidate nuclease and to find the window between under and overexposure of the test molecule to the polymerase preparation beinginvestigated.

The above described modified polymerases were tested for 5′ nucleaseactivity as follows: Reaction 1 was performed in a buffer of 10 mMTris-Cl, pH 8.5 at 20° C., 1.5 mM MgCl₂ and 50 mM KCl and in Reaction 2the KCl concentration was reduced to 20 mM. In Reactions 1 and 2, 10fmoles of the test substrate molecule shown in FIG. 16E were combinedwith 1 pmole of the indicated primer and 0.5 to 1.0 μl of extractcontaining the modified polymerase (prepared as described above). Thismixture was then incubated for 10 minutes at 55° C. For all of themutant polymerases tested these conditions were sufficient to givecomplete cleavage. When the molecule shown in FIG. 16E was labeled atthe 5′ end, the released 5′ fragment, 25 nucleotides long, wasconveniently resolved on a 20% polyacrylamide gel (19:1 cross-linked)with 7 M urea in a buffer containing 45 mM Tris-borate pH 8.3, 1.4 mMEDTA. Clones 4C—F and 5B exhibited structure-specific cleavagecomparable to that of the unmodified DNA polymerase. Additionally,clones 4E, 4F and 4G have the added ability to cleave DNA in the absenceof a 3′ arm as discussed above. Representative cleavage reactions areshown in FIG. 17.

For the reactions shown in FIG. 17, the mutant polymerase clones 4E (Taqmutant) and 5B (Tfl mutant) were examined for their ability to cleavethe hairpin substrate molecule shown in FIG. 16E. The substrate moleculewas labeled at the 5′ terminus with ³²P. Ten fmoles of heat-denatured,end-labeled substrate DNA and 0.5 units of DNAPTaq (lane 1) or 0.5 μl of4e or 5b extract (FIG. 17, lanes 2-7, extract was prepared as describedabove) were mixed together in a buffer containing 10 mM Tris-Cl, pH 8.5,50 mM KCl and 1.5 mM MgCl₂. The final reaction volume was 10 μl.Reactions shown in lanes 4 and 7 contain in addition 50 μM of each dNTP.Reactions shown in lanes 3, 4, 6 and 7 contain 0.2 μM of the primeroligonucleotide (complementary to the 3′ arm of the substrate and shownin FIG. 16E). Reactions were incubated at 55° C. for 4 minutes.Reactions were stopped by the addition of 8 μl of 95% formamidecontaining 20 mM EDTA and 0.05% marker dyes per 10 μl reaction volume.Samples were then applied to 12% denaturing acrylamide gels. Followingelectrophoresis, the gels were autoradiographed. FIG. 17 shows thatclones 4E and 5B exhibit cleavage activity similar to that of the nativeDNAPTaq. Note that some cleavage occurs in these reactions in theabsence of the primer. When long hairpin structure, such as the one usedhere (FIG. 16E), are used in cleavage reactions performed in bufferscontaining 50 mM KCl a low level of primer-independent cleavage is seen.Higher concentrations of KCl suppress, but do not eliminate, thisprimer-independent cleavage under these conditions.

2. Assay For Synthetic Activity

The ability of the modified enzyme or proteolytic fragments is assayedby adding the modified enzyme to an assay system in which a primer isannealed to a template and DNA synthesis is catalyzed by the addedenzyme. Many standard laboratory techniques employ such an assay. Forexample, nick translation and enzymatic sequencing involve extension ofa primer along a DNA template by a polymerase molecule.

In a preferred assay for determining the synthetic activity of amodified enzyme an oligonucleotide primer is annealed to asingle-stranded DNA template, e.g., bacteriophage M13 DNA, and theprimer/template duplex is incubated in the presence of the modifiedpolymerase in question, deoxynucleoside triphosphates (dNTPs) and thebuffer and salts known to be appropriate for the unmodified or nativeenzyme. Detection of either primer extension (by denaturing gelelectrophoresis) or dNTP incorporation (by acid precipitation orchromatography) is indicative of an active polymerase. A label, eitherisotopic or non-isotopic, is preferably included on either the primer oras a dNTP to facilitate detection of polymerization products. Syntheticactivity is quantified as the amount of free nucleotide incorporatedinto the growing DNA chain and is expressed as amount incorporated perunit of time under specific reaction conditions.

Representative results of an assay for synthetic activity is shown inFIG. 18. The synthetic activity of the mutant DNAPTaq clones 4B-F wastested as follows: A master mixture of the following buffer was made:1.2×PCR buffer (1×PCR buffer contains 50 mM KCl, 1.5 mM MgCl₂, 10 mMTris-Cl, ph 8.5 and 0.05% each TWEEN 20 and NONIDET P40), 50 μM each ofdGTP, dATP and dTTP, 5 μM dCTP and 0.125 μM α-³²P-dCTP at 600 Ci/mmol.Before adjusting this mixture to its final volume, it was divided intotwo equal aliquots. One received distilled water up to a volume of 50 μlto give the concentrations above. The other received 5 μg ofsingle-stranded M13 mp18 DNA (approximately 2.5 pmol or 0.05 μM finalconcentration) and 250 pmol of M13 sequencing primer (5 μM finalconcentration) and distilled water to a final volume of 50 μl. Eachcocktail was warmed to 75° C. for 5 minutes and then cooled to roomtemperature. This allowed the primers to anneal to the DNA in theDNA-containing mixtures.

For each assay, 4 μl of the cocktail with the DNA was combined with 1 μlof the mutant polymerase, prepared as described, or 1 unit of DNAPTaq(Perkin Elmer) in 1 μl of dH₂O. A “no DNA” control was done in thepresence of the DNAPTaq (FIG. 18, lane 1), and a “no enzyme” control wasdone using water in place of the enzyme (lane 2). Each reaction wasmixed, then incubated at room temperature (approx. 22° C.) for 5minutes, then at 55° C. for 2 minutes, then at 72° C. for 2 minutes.This step incubation was done to detect polymerization in any mutantsthat might have optimal temperatures lower than 72° C. After the finalincubation, the tubes were spun briefly to collect any condensation andwere placed on ice. One μl of each reaction was spotted at an origin 1.5cm from the bottom edge of a polyethyleneimine (PEI) cellulose thinlayer chromatography plate and allowed to dry. The chromatography platewas run in 0.75 M NaH₂PO₄, pH 3.5, until the buffer front had runapproximately 9 cm from the origin. The plate was dried, wrapped inplastic wrap, marked with luminescent ink, and exposed to X-ray film.Incorporation was detected as counts that stuck where originallyspotted, while the unincorporated nucleotides were carried by the saltsolution from the origin.

Comparison of the locations of the counts with the two control lanesconfirmed the lack of polymerization activity in the mutantpreparations. Among the modified DNAPTaq clones, only clone 4B retainsany residual synthetic activity as shown in FIG. 18.

Example 3 5′ Nucleases Derived from Thermostable DNA Polymerases canCleave Short Hairpin Structures with Specificity

The ability of the 5′ nucleases to cleave hairpin structures to generatea cleaved hairpin structure suitable as a detection molecule wasexamined. The structure and sequence of the hairpin test molecule isshown in FIG. 19A (SEQ ID NO:15). The oligonucleotide (labeled “primer”in FIG. 19A, SEQ ID NO:22) is shown annealed to its complementarysequence on the 3′ arm of the hairpin test molecule. The hairpin testmolecule was single-end labeled with ³²P using a labeled T7 promoterprimer in a polymerase chain reaction. The label is present on the 5′arm of the hairpin test molecule and is represented by the star in FIG.19A.

The cleavage reaction was performed by adding 10 fmoles ofheat-denatured, end-labeled hairpin test molecule, 0.2 uM of the primeroligonucleotide (complementary to the 3′ arm of the hairpin), 50 μM ofeach dNTP and 0.5 units of DNAPTaq (Perkin Elmer) or 0.5 μl of extractcontaining a 5′ nuclease (prepared as described above) in a total volumeof 10 μl in a buffer containing 10 mM Tris-Cl, pH 8.5, 50 mM KCl and 1.5mM MgCl₂. Reactions shown in lanes 3, 5 and 7 were run in the absence ofdNTPs.

Reactions were incubated at 55° C. for 4 minutes. Reactions were stoppedat 55° C. by the addition of 8 μl of 95% formamide with 20 mM EDTA and0.05% marker dyes per 10 μl reaction volume. Samples were not heatedbefore loading onto denaturing polyacrylamide gels (10% polyacrylamide,19:1 crosslinking, 7 M urea, 89 mM Tris-borate, pH 8.3, 2.8 mM EDTA).The samples were not heated to allow for the resolution ofsingle-stranded and re-duplexed uncleaved hairpin molecules.

FIG. 19B shows that altered polymerases lacking any detectable syntheticactivity cleave a hairpin structure when an oligonucleotide is annealedto the single-stranded 3′ arm of the hairpin to yield a single speciesof cleaved product (FIG. 19B, lanes 3 and 4). 5′ nucleases, such asclone 4D, shown in lanes 3 and 4, produce a single cleaved product evenin the presence of dNTPs. 5′ nucleases which retain a residual amount ofsynthetic activity (less than 1% of wild type activity) produce multiplecleavage products as the polymerase can extend the oligonucleotideannealed to the 3′ arm of the hairpin thereby moving the site ofcleavage (clone 4B, lanes 5 and 6). Native DNATaq produces even morespecies of cleavage products than do mutant polymerases retainingresidual synthetic activity and additionally converts the hairpinstructure to a double-stranded form in the presence of dNTPs due to thehigh level of synthetic activity in the native polymerase (FIG. 19B,lane 8).

Example 4 Test of the Trigger/Detection Assay

To test the ability of an oligonucleotide of the type released in thetrigger reaction of the trigger/detection assay to be detected in thedetection reaction of the assay, the two hairpin structures shown inFIG. 20A were synthesized using standard techniques. The two hairpinsare termed the A-hairpin (SEQ ID NO:23) and the T-hairpin (SEQ IDNO:24). The predicted sites of cleavage in the presence of theappropriate annealed primers are indicated by the arrows. The A- andT-hairpins were designed to prevent intra-strand mis-folding by omittingmost of the T residues in the A-hairpin and omitting most of the Aresidues in the T-hairpin. To avoid mis-priming and slippage, thehairpins were designed with local variations in the sequence motifs(e.g., spacing T residues one or two nucleotides apart or in pairs). TheA- and T-hairpins can be annealed together to form a duplex which hasappropriate ends for directional cloning in pUC-type vectors;restriction sites are located in the loop regions of the duplex and canbe used to elongate the stem regions if desired.

The sequence of the test trigger oligonucleotide is shown in FIG. 20B;this oligonucleotide is termed the alpha primer (SEQ ID NO:25). Thealpha primer is complementary to the 3′ arm of the T-hairpin as shown inFIG. 20A. When the alpha primer is annealed to the T-hairpin, a cleavagestructure is formed that is recognized by thermostable DNA polymerases.Cleavage of the T-hairpin liberates the 5′ single-stranded arm of theT-hairpin, generating the tau primer (SEQ ID NO:26) and a cleavedT-hairpin (FIG. 20B; SEQ ID NO:27). The tau primer is complementary tothe 3′ arm of the A-hairpin as shown in FIG. 20A. Annealing of the tauprimer to the A-hairpin generates another cleavage structure; cleavageof this second cleavage structure liberates the 5′ single-stranded armof the A-hairpin, generating another molecule of the alpha primer whichthen is annealed to another molecule of the T-hairpin. Thermocyclingreleases the primers so they can function in additional cleavagereactions. Multiple cycles of annealing and cleavage are carried out.The products of the cleavage reactions are primers and the shortenedhairpin structures shown in FIG. 20C. The shortened or cleaved hairpinstructures may be resolved from the uncleaved hairpins byelectrophoresis on denaturing acrylamide gels.

The annealing and cleavage reactions are carried as follows: In a 50 μlreaction volume containing 10 mM Tris-Cl, pH 8.5, 1.0 MgCl₂, 75 mM KCl,1 pmole of A-hairpin, 1 pmole T-hairpin, the alpha primer is added atequimolar amount relative to the hairpin structures (1 pmole) or atdilutions ranging from 10- to 10⁶-fold and 0.5 μl of extract containinga 5′ nuclease (prepared as described above) are added. The predictedmelting temperature for the alpha or trigger primer is 60° C. in theabove buffer. Annealing is performed just below this predicted meltingtemperature at 55° C. Using a Perkin Elmer DNA Thermal Cycler, thereactions are annealed at 55° C. for 30 seconds. The temperature is thenincreased slowly over a five minute period to 72° C. to allow forcleavage. After cleavage, the reactions are rapidly brought to 55° C.(1° C. per second) to allow another cycle of annealing to occur. A rangeof cycles are performed (20, 40 and 60 cycles) and the reaction productsare analyzed at each of these number of cycles. The number of cycleswhich indicates that the accumulation of cleaved hairpin products hasnot reached a plateau is then used for subsequent determinations when itis desirable to obtain a quantitative result.

Following the desired number of cycles, the reactions are stopped at 55°C. by the addition of 8 μl of 95% formamide with 20 mM EDTA and 0.05%marker dyes per 10 μl reaction volume. Samples are not heated beforeloading onto denaturing polyacrylamide gels (10% polyacrylamide, 19:1crosslinking, 7 M urea, 89 mM tris-borate, pH 8.3, 2.8 mM EDTA). Thesamples were not heated to allow for the resolution of single-strandedand re-duplexed uncleaved hairpin molecules.

The hairpin molecules may be attached to separate solid supportmolecules, such as agarose, styrene or magnetic beads, via the 3′ end ofeach hairpin. A spacer molecule may be placed between the 3′ end of thehairpin and the bead if so desired. The advantage of attaching thehairpins to a solid support is that this prevents the hybridization ofthe A- and T-hairpins to one another during the cycles of melting andannealing. The A- and T-hairpins are complementary to one another (asshown in FIG. 20D) and if allowed to anneal to one another over theirentire lengths this would reduce the amount of hairpins available forhybridization to the alpha and tau primers during the detectionreaction.

The 5′ nucleases of the present invention are used in this assay becausethey lack significant synthetic activity. The lack of synthetic activityresults in the production of a single cleaved hairpin product (as shownin FIG. 19B, lane 4). Multiple cleavage products may be generated by 1)the presence of interfering synthetic activity (see FIG. 19B, lanes 6and 8) or 2) the presence of primer-independent cleavage in thereaction. The presence of primer-independent cleavage is detected in thetrigger/detection assay by the presence of different sized products atthe fork of the cleavage structure. Primer-independent cleavage can bedampened or repressed, when present, by the use of uncleavablenucleotides in the fork region of the hairpin molecule. For example,thiolated nucleotides can be used to replace several nucleotides at thefork region to prevent primer-independent cleavage.

Example 5 Cleavage of Linear Nucleic Acid Substrates

From the above, it should be clear that native (i.e., “wild type”)thermostable DNA polymerases are capable of cleaving hairpin structuresin a specific manner and that this discovery can be applied with successto a detection assay. In this example, the mutant DNAPs of the presentinvention are tested against three different cleavage structures shownin FIG. 22A. Structure 1 in FIG. 22A is simply single stranded 206-mer(the preparation and sequence information for which was discussedabove). Structures 2 and 3 are duplexes; structure 2 is the same hairpinstructure as shown in FIG. 12A (bottom), while structure 3 has thehairpin portion of structure 2 removed.

The cleavage reactions comprised 0.01 pmoles of the resulting substrateDNA, and 1 pmole of pilot oligonucleotide in a total volume of 10 μl of10 mM Tris-Cl, pH 8.3, 100 mM KCl, 1 mM MgCl₂. Reactions were incubatedfor 30 minutes at 55° C., and stopped by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to75° C. for 2 minutes immediately before electrophoresis through a 10%polyacrylamide gel (19:1 cross link), with 7M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA.

The results were visualized by autoradiography and are shown in FIG. 22Bwith the enzymes indicated as follows: I is native Taq DNAP; II isnative Tfl DNAP; III is the enzyme CLEAVASE BX shown in FIG. 4E; IV isthe enzyme CLEAVASE BB shown in FIG. 4F; V is the mutant shown in FIG.5B; and VI is the enzyme CLEAVASE BN shown in FIG. 4G. Structure 2 wasused to “normalize” the comparison. For example, it was found that ittook 50 ng of Taq DNAP and 300 ng of the enzyme CLEAVASE BN to givesimilar amounts of cleavage of Structure 2 in thirty (30) minutes. Underthese conditions native Taq DNAP is unable to cleave Structure 3 to anysignificant degree. Native Tfl DNAP cleaves Structure 3 in a manner thatcreates multiple products.

By contrast, all of the mutants tested cleave the linear duplex ofStructure 3. This finding indicates that this characteristic of themutant DNA polymerases is consistent of thermostable polymerases acrossthermophilic species.

The finding described herein that the mutant DNA polymerases of thepresent invention are capable of cleaving linear duplex structuresallows for application to a more straightforward assay design (FIG. 1A).FIG. 23 provides a more detailed schematic corresponding to the assaydesign of FIG. 1A.

The two 43-mers depicted in FIG. 23 were synthesized by standardmethods. Each included a fluorescein on the 5′ end for detectionpurposes and a biotin on the 3′ end to allow attachment to streptavidincoated paramagnetic particles (the biotin-avidin attachment is indicatedby “ ”).

Before the trityl groups were removed, the oligos were purified by HPLCto remove truncated by-products of the synthesis reaction. Aliquots ofeach 43-mer were bound to M-280 DYNABEADS (Dynal) at a density of 100pmoles per mg of beads. Two (2) mgs of beads (200 μl) were washed twicein 1× wash/bind buffer (1 M NaCl, 5 mM Tris-Cl, pH 7.5, 0.5 mM EDTA)with 0.1% BSA, 200 μl per wash. The beads were magnetically sedimentedbetween washes to allow supernatant removal. After the second wash, thebeads were resuspended in 200 μl of 2× wash/bind buffer (2 M Na Cl, 10mM Tris-Cl, pH 7.5 with 1 mM EDTA), and divided into two 100 μlaliquots. Each aliquot received 1 μl of a 100 μM solution of one of thetwo oligonucleotides. After mixing, the beads were incubated at roomtemperature for 60 minutes with occasional gentle mixing. The beads werethen sedimented and analysis of the supernatants showed only traceamounts of unbound oligonucleotide, indicating successful binding. Eachaliquot of beads was washed three times, 100 μl per wash, with 1×wash/bind buffer, then twice in a buffer of 10 mM Tris-Cl, pH 8.3 and 75mM KCl. The beads were resuspended in a final volume of 100 μl of theTris/KCl, for a concentration of 1 pmole of oligo bound to 10 μg ofbeads per μl of suspension. The beads were stored at 4° C. between uses.

The types of beads correspond to FIG. 1A. That is to say, type 2 beadscontain the oligo (SEQ ID NO:33) comprising the complementary sequence(SEQ ID NO:34) for the alpha signal oligo (SEQ ID NO:35) as well as thebeta signal oligo (SEQ ID NO:36) which when liberated is a 24-mer. Thisoligo has no “As” and is “T” rich. Type 3 beads contain the oligo (SEQID NO:37) comprising the complementary sequence (SEQ ID NO:38) for thebeta signal oligo (SEQ ID NO:39) as well as the alpha signal oligo (SEQID NO:35) which when liberated is a 20-mer. This oligo has no “Ts” andis “A” rich.

Cleavage reactions comprised 1 μl of the indicated beads, 10 pmoles ofunlabelled alpha signal oligo as “pilot” (if indicated) and 500 ng ofthe enzyme CLEAVASE BN in 20 μl of 75 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.5mM MgCl₂ and 10 μM CTAB. All components except the enzyme wereassembled, overlaid with light mineral oil and warmed to 53° C. Thereactions were initiated by the addition of prewarmed enzyme andincubated at that temperature for 30 minutes. Reactions were stopped attemperature by the addition of 16 μl of 95% formamide with 20 mM EDTAand 0.05% each of bromophenol blue and xylene cyanol. This additionstops the enzyme activity and, upon heating, disrupts the biotin-avidinlink, releasing the majority (greater than 95%) of the oligos from thebeads. Samples were heated to 75° C. for 2 minutes immediately beforeelectrophoresis through a 10% polyacrylamide gel (19:1 cross link), with7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Resultswere visualized by contact transfer of the resolved DNA to positivelycharged nylon membrane and probing of the blocked membrane with ananti-fluorescein antibody conjugated to alkaline phosphatase. Afterwashing, the signal was developed by incubating the membrane in WESTERNBLUE (Promega) which deposits a purple precipitate where the antibody isbound.

FIG. 24 shows the propagation of cleavage of the linear duplex nucleicacid structures of FIG. 23 by the DNAP mutants of the present invention.The two center lanes contain both types of beads. As noted above, thebeta signal oligo (SEQ ID NO:36) when liberated is a 24-mer and thealpha signal oligo (SEQ ID NO:35) when liberated is a 20-mer. Theformation of the two lower bands corresponding to the 24-mer and 20-meris clearly dependent on “pilot”.

Example 6 5′ Exonucleolytic Cleavage (“Nibbling”) by Thermostable DNAPs

It has been found that thermostable DNAPs, including those of thepresent invention, have a true 5′ exonuclease capable of nibbling the 5′end of a linear duplex nucleic acid structures. In this example, the 206base pair DNA duplex substrate is again employed (see above). In thiscase, it was produced by the use of one ³²P-labeled primer and oneunlabeled primer in a polymerase chain reaction. The cleavage reactionscomprised 0.01 pmoles of heat-denatured, end-labeled substrate DNA (withthe unlabeled strand also present), 5 pmoles of pilot oligonucleotide(see pilot oligos in FIG. 12A) and 0.5 units of DNAPTaq or 0.5μ of theenzyme CLEAVASE BB in the E. coli extract (see above), in a total volumeof 10 μl of 10 mM Tris.Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂.

Reactions were initiated at 65° C. by the addition of pre-warmed enzyme,then shifted to the final incubation temperature for 30 minutes. Theresults are shown in FIG. 25A. Samples in lanes 1-4 are the results withnative Tag DNAP, while lanes 5-8 shown the results with the enzymeCLEAVASE BB. The reactions for lanes 1, 2, 5, and 6 were performed at65° C. and reactions for lanes 3, 4, 7, and 8 were performed at 50° C.and all were stopped at temperature by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to75° C. for 2 minutes immediately before electrophoresis through a 10%acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mMTris.Borate, pH 8.3, 1.4 mM EDTA. The expected product in reactions 1,2, 5, and 6 is 85 nucleotides long; in reactions 3 and 7, the expectedproduct is 27 nucleotides long. Reactions 4 and 8 were performed withoutpilot, and should remain at 206 nucleotides. The faint band seen at 24nucleotides is residual end-labeled primer from the PCR.

The surprising result is that the enzyme CLEAVASE BB under theseconditions causes all of the label to appear in a very small species,suggesting the possibility that the enzyme completely hydrolyzed thesubstrate. To determine the composition of the fastest-migrating bandseen in lanes 5-8 (reactions performed with the deletion mutant),samples of the 206 base pair duplex were treated with either T7 gene 6exonuclease (USB) or with calf intestine alkaline phosphatase (Promega),according to manufacturers' instructions, to produce either labeledmononucleotide (lane a of FIG. 25B) or free ³²P-labeled inorganicphosphate (lane b of FIG. 25B), respectively. These products, along withthe products seen in lane 7 of panel A were resolved by briefelectrophoresis through a 20% acrylamide gel (19:1 cross-link), with 7 Murea, in a buffer of 45 mM Tris.Borate, pH 8.3, 1.4 mM EDTA. The enzymeCLEAVASE BB is thus capable of converting the substrate tomononucleotides.

Example 7 Nibbling is Duplex Dependent

The nibbling by the enzyme CLEAVASE BB is duplex dependent. In thisexample, internally labeled, single strands of the 206-mer were producedby 15 cycles of primer extension incorporating α-³²P labeled dCTPcombined with all four unlabeled dNTPs, using an unlabeled 206-bpfragment as a template. Single and double stranded products wereresolved by electrophoresis through a non-denaturing 6%polyacrylamide:gel (29:1 cross-link) in a buffer of 45 mM Tris.Borate,pH 8.3, 1.4 mM EDTA, visualized by autoradiography, excised from thegel, eluted by passive diffusion, and concentrated by ethanolprecipitation.

The cleavage reactions comprised 0.04 pmoles of substrate DNA, and 2 μlof the enzyme CLEAVASE BB (in an E. coli extract as described above) ina total volume of 40 μl of 10 mM Tris.Cl, pH 8.5, 50 mM KCl, 1.5 mMMgCl₂. Reactions were initiated by the addition of pre-warmed enzyme; 10μl aliquots were removed at 5, 10, 20, and 30 minutes, and transferredto prepared tubes containing 8 μl of 95% formamide with 30 mM EDTA and0.05% marker dyes. Samples were heated to 75° C. for 2 minutesimmediately before electrophoresis through a 10% acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris.Borate, pH 8.3,1.4 mM EDTA. Results were visualized by autoradiography as shown in FIG.26. Clearly, the cleavage by the enzyme CLEAVASE BB depends on a duplexstructure; no cleavage of the single strand structure is detectedwhereas cleavage of the 206-mer duplex is complete.

Example 8 Nibbling can be Target Directed

The nibbling activity of the DNAPs of the present invention can beemployed with success in a detection assay. One embodiment of such anassay is shown in FIG. 27. In this assay, a labelled oligo is employedthat is specific for a target sequence. The oligo is in excess of thetarget so that hybridization is rapid. In this embodiment, the oligocontains two fluorescein labels whose proximity on the oligo causestheir emission to be quenched. When the DNAP is permitted to nibble theoligo the labels separate and are detectable. The shortened duplex isdestabilized and disassociates. Importantly, the target is now free toreact with an intact labelled oligo. The reaction can continue until thedesired level of detection is achieved. An analogous, althoughdifferent, type of cycling assay has been described employing lambdaexonuclease. See C. G. Copley and C. Boot, BioTechniques 13:888 (1992).

The success of such an assay depends on specificity. In other words, theoligo must hybridize to the specific target. It is also preferred thatthe assay be sensitive; the oligo ideally should be able to detect smallamounts of target. FIG. 28A shows a 5′-end ³²P-labelled primer bound toa plasmid target sequence. In this case, the plasmid was pUC19(commercially available) which was heat denatured by boiling two (2)minutes and then quick chilling. The primer is a 21-mer (SEQ ID NO:39).The enzyme CLEAVASE BX (a dilution equivalent to 5×10⁻³ μl extract) wasemployed in 100 mM KCl, 10 mM Tris-Cl, pH 8.3, 2 mM MnCl₂. The reactionwas performed at 55° C. for sixteen (16) hours with or without genomicbackground DNA (from chicken blood). The reaction was stopped by theaddition of 8 μl of 95% formamide with 20 mM EDTA and marker dyes.

The products of the reaction were resolved by PAGE (10% polyacrylamide,19:1 cross link, 1×TBE) as seen in FIG. 28B. Lane “M” contains thelabelled 21-mer. Lanes 1-3 contain no specific target, although Lanes 2and 3 contain 100 ng and 200 ng of genomic DNA, respectively. Lanes 4, 5and 6 all contain specific target with either 0 ng, 100 ng or 200 ng ofgenomic DNA, respectively. It is clear that conversion tomononucleotides occurs in Lanes 4, 5 and 6 regardless of the presence oramount of background DNA. Thus, the nibbling can be target directed andspecific.

Example 9 Purification of the Enzyme Cleavase

As noted above, expressed thermostable proteins, i.e., the 5′ nucleases,were isolated by crude bacterial cell extracts. The precipitated E. coliproteins were then, along with other cell debris, removed bycentrifugation. In this example, cells expressing the BN clone werecultured and collected (500 grams). For each gram (wet weight) of E.coli, 3 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 μMNaCl) was added. The cells were lysed with 200 μg/ml lysozyme at roomtemperature for 20 minutes. Thereafter deoxycholic acid was added tomake a 0.2% final concentration and the mixture was incubated 15 minutesat room temperature.

The lysate was sonicated for approximately 6-8 minutes at 0° C. Theprecipitate was removed by centrifugation (39,000 g for 20 minutes).Polyethyleneimine was added (0.5%) to the supernatant and the mixturewas incubated on ice for 15 minutes. The mixture was centrifuged (5,000g for 15 minutes) and the supernatant was retained. This was heated for30 minutes at 60° C. and then centrifuged again (5,000 g for 15 minutes)and the supernatant was again retained.

The supernatant was precipitated with 35% ammonium sulfate at 4° C. for15 minutes. The mixture was then centrifuged (5,000 g for 15 minutes)and the supernatant was removed. The precipitate was then dissolved in0.25 M KCl, 20 mM Tris, pH 7.6, 0.2% TWEEN and 0.1 EDTA) and thendialyzed against Binding Buffer (8× Binding Buffer comprises: 40 mMimidazole, 4M NaCl, 160 mM Tris-HCl, pH 7.9).

The solubilized protein is then purified on the Ni⁺⁺ column (Novagen).The Binding Buffer is allows to drain to the top of the column bed andload the column with the prepared extract. A flow rate of about 10column volumes per hour is optimal for efficient purification. If theflow rate is too fast, more impurities will contaminate the elutedfraction.

The column is washed with 25 ml (10 volumes) of 1× Binding Buffer andthen washed with 15 ml (6 volumes) of 1× Wash Buffer (8× Wash Buffercomprises: 480 mM imidazole, 4M NaCl, 160 mM Tris-HCl, pH 7.9). Thebound protein was eluted with 15 ml (6 volumes) of 1× Elute Buffer (4×Elute Buffer comprises: 4 mM imidazole, 2M NaCl, 80 mM Tris-HCl, pH7.9). Protein is then reprecipitated with 35% Ammonium Sulfate as above.The precipitate was then dissolved and dialyzed against: 20 mM Tris, 100mM KCl, 1 mM EDTA). The solution was brought up to 0.1% each of TWEEN 20and NP-40 and stored at 4° C.

Example 10 5′ Nucleases Cut Nucleic Acid Substrates at NaturallyOccurring Areas of Secondary Structure

The ability of a 5′ nuclease to recognize and cleave nucleic acidsubstrates at naturally occurring areas of secondary structure in theabsence of a pilot oligonucleotide (i.e., primer independent cleavage)was shown in Example 1C (FIG. 12, lane 9). When DNAPTaq was incubated at50° C. in the presence of a 206 by DNA substrate (single end labeled,double stranded template) in a buffer containing 10 mM Tris-HCl, pH 8.5and 1.5 mM MgCl₂, adventitious (i.e., naturally occurring) structures inthe DNA substrate were cleaved by the 5′ nuclease activity of theenzyme. This cleavage generated three prominent fragments (FIG. 12, lane9); this cleavage pattern provides a “fingerprint” of the DNA template.

The ability of 5′ nucleases to cleave naturally occurring structures innucleic acid templates (structure-specific cleavage) is useful to detectinternal sequence differences in nucleic acids without prior knowledgeof the specific sequence of the nucleic acid. To develop a generalmethod to scan nucleic acids for mutations (e.g., single base changes(point mutations), small insertions or deletions, etc.) using 5′nucleases, the following series of experiments were performed.

A. The Substitution of MnCl₂ for MgCl₂ in the Cleavage Reaction ProducesEnhanced Cleavage Patterns

The effect of substituting of Mn²⁺ in place of Mg²⁺ upon the cleavagepattern created by 5′ nuclease activity on a double-stranded DNAsubstrate was examined. A 157 by fragment derived from exon 4 of eitherthe wild-type (SEQ ID NO:40) or the mutant G419R (SEQ ID NO:41)tyrosinase gene was prepared by PCR as follows.

The primer pair 5′ biotin-CACCGTCCTCTTCAAGAAG 3′ (SEQ ID NO:42) and 5′fluorescein-CTGAATCTTGTAGATAGCTA 3′ (SEQ ID NO:43) was used to prime thePCRs. The synthetic primers were obtained from Promega; the primers werelabeled on the 5′ end with biotin or fluorescein during synthesis.

The target DNA for the generation of the 157 by fragment of mutant G419R(King, R. A., et al., (1991) Mol. Biol. Med. 8:19; here after referredto as the 419 mutant) was a 339 by PCR product (SEQ ID NO:44) generatedusing genomic DNA homozygous for the 419 mutation. Genomic DNA wasisolated using standard techniques from peripheral blood leukocytesisolated from patients. This 339 by PCR product was prepared as follows.

The symmetric PCR reaction comprised 10 ng of genomic DNA from the 419mutant, 100 pmoles of the primer 5′ biotin-GCCTTATTTTACTTTAAAAAT-3′ (SEQID NO:45), 100 pmoles of the primer 5′fluorescein-TAAAGTTTTGTGTTATCTCA-3′ (SEQ ID NO:46), 50 μM of each dNTP,20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and0.05% NONIDET P-40 (NP40). The primers of SEQ ID NOS:45 and 46 wereobtained from Integrated DNA Technologies, Coralville, IA. A tubecontaining 45 μl of the above mixture was overlaid with two drops oflight mineral oil and the tube was heated to 95° C. for 1 min. Taqpolymerase was then added as 1.25 units of enzyme in 5 μl of 20 mMTris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05%NONIDET P-40. The tube was heated to 94° C. for 40 sec, cooled to 55° C.for 50 sec, heated to 72° C. for 70 sec for 29 repetitions with a 5 minincubation at 72° C. after the last repetition.

The PCR products were gel purified as follows. The products wereresolved by electrophoresis through a 6% polyacrylamide gel (29:1cross-link) in a buffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mMEDTA. The DNA was visualized by ethidium bromide staining and the 339 byfragment was excised from the gel. The DNA was eluted from the gel sliceby passive diffusion overnight into a solution containing 0.5 M NH₄OAc,0.1% SDS and 0.1 M EDTA. The DNA was then precipitated with ethanol inthe presence of 4 μg of glycogen carrier. The DNA was pelleted andresuspended in 40 μl of TE (10 mM Tris-Cl, pH 8.0, 0.1 mM EDTA).

To generate the 157 by fragment from the 419 mutant, the purified 339 by419 PCR fragment was used as the target in an asymmetric PCR. Theasymmetric PCR comprised 100 pmoles of the biotinylated primer of SEQ IDNO:45, 1 pmole of the fluoresceinated primer of SEQ ID NO:46, 50 μM ofeach dNTP, 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05%TWEEN 20 and 0.05% NONIDET P-40. A tube containing 45 μl of the abovemixture was overlaid with two drops of light mineral oil and the tubewas heated to 95° C. for 5 sec and then cooled to 70° C. Taq polymerasewas then added as 1.25 units of enzyme in 5 μl of 20 mM Tris-Cl, pH 8.3,1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05% NONIDET P-40. Thetube was heated to 95° C. for 45 sec, cooled to 50° C. for 45 sec,heated to 72° C. for 1 min 15 sec for 30 repetitions with a 5 minincubation at 72° C. after the last repetition.

The asymmetric PCR products were gel purified as follows. The productswere resolved by electrophoresis through a 6% polyacrylamide gel (29:1cross-link) in a buffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mMEDTA. The DNA was visualized by ethidium bromide staining; thedouble-stranded DNA was differentiated from the single-stranded DNA dueto the mobility shift commonly seen with single-stranded DNA producedfrom asymmetric PCR (In an asymmetric PCR both single-stranded anddouble-stranded products are produced; typically the single-strandedproduct will have a slower speed of migration through the gel and willappear closer to the origin than will the double-stranded product). Thedouble-stranded 157 by substrate corresponding to the 419 mutant (SEQ IDNO:41) was excised from the gel.

The 157 by wild-type fragment was generated by asymmetric PCR asdescribed above for the 419 mutant with the exception that the targetDNA was 10 ng of supercoiled pcTYR-N1Tyr plasmid DNA. The pcTYR-N1Tyrplasmid contains the entire wild-type tyrosinase cDNA (Geibel, L. B., etal. (1991) Genomics 9:435).

Following the asymmetric PCRs, the reaction products were resolved on anacrylamide gel and the double-stranded fragments of interest wereexcised, eluted and precipitated as described above. The precipitated157 by wild-type (SEQ ID NO:40) and 419 mutant (SEQ ID NO:41) fragmentswere resuspended in 40 μl of TE.

Cleavage reactions comprised 100 fmoles of the resulting double-strandedsubstrate DNAs (the substrates contain a biotin moiety at the 5′ end ofthe sense strand) in a total volume of 10 μl of 10 mM MOPS, pH 8.2, 1 mMdivalent cation (either MgCl₂ or MnCl₂) and 1 unit of DNAPTaq. Thereactions were overlaid with a drop of light mineral oil. Reactions wereheated to 95° C. for 5 seconds to denature the substrate and then thetubes were quickly cooled to 65° C. (this step allows the DNA assume itsunique secondary structure by allowing the formation of intra-strandhydrogen bonds between complimentary bases). The reaction can beperformed in either a thermocycler (MJ Research, Watertown, Mass.)programmed to heat to 95° C. for 5 seconds then drop the temperatureimmediately to 65° C. or alternatively the tubes can be placed manuallyin a heat block set at 95° C. and then transferred to a second heatblock set at 65° C.

The reaction was incubated at 65° C. for 10 minutes and was stopped bythe addition of 8 μl of stop buffer (95% formamide containing 20 mM EDTAand 0.05% each xylene cyanol and bromophenol blue). Samples were heatedto 72° C. for 2 minutes and 5 μl of each reaction were resolved byelectrophoresis through a 10% polyacrylamide gel (19:1 cross-link), with7 M urea, in a buffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated allowing the gel toremain flat on one plate. A 0.2 μm-pore positively-charged nylonmembrane (Schleicher and Schuell, Keene, N.H.), pre-wetted in 0.5×TBE(45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA), was laid on top of the exposedacrylamide gel. All air bubbles trapped between the gel and the membranewere removed. Two pieces of 3MM filter paper (Whatman) were then placedon top of the membrane, the other glass plate was replaced, and thesandwich was clamped with binder clips. Transfer was allowed to proceedovernight. After transfer, the membrane was carefully peeled from thegel and allowed to air dry. After complete drying, the membrane waswashed in 1.2× SEQUENASE IMAGES Blocking Buffer (United StatesBiochemical) for 30 minutes. Three tenths of a ml of the buffer was usedper cm² of membrane. A streptavidin-alkaline phosphatase conjugate(SAAP, United States Biochemical) was added to a 1:4000 dilutiondirectly to the blocking solution, and agitated for 15 minutes. Themembrane was rinsed briefly with H₂O and then washed 3 times (5minutes/wash) in 1×SAAP buffer (100 mM Tris-HCL, pH 10; 50 mM NaCl) with0.1% sodium dodecyl sulfate (SDS) using 0.5 ml buffer/cm² of the buffer,with brief H₂O rinses between each wash. Similarly, forfluorescein-labeled DNA, anti-fluorescein fragment (Boehringer MannheimBiochemicals, Indianapolis, Ind.) at a 1:20,000 final dilution may beadded followed by three washes (5 min/wash) in 1×SAAP buffer containing0.1% SDS and 0.025% TWEEN 20. The membrane was then washed once in1×SAAP buffer without SDS, drained thoroughly and placed in a plasticheat-sealable bag. Using a sterile pipet tip, 0.05 ml/cm² of CDPSTARreagent (Tropix, Bedford, Mass.) was added to the bag and distributedover the entire membrane for 5 minutes. The bag was drained of allexcess liquid and air bubbles. The membrane was then exposed to X-rayfilm (Kodax XRP) for an initial 30 minutes. Exposure times were adjustedas necessary for resolution and clarity. The results are shown in FIG.30.

In FIG. 30, the lane marked “M” contains molecular weight markers. Themarker fragments were generated by digestion of pUC19 with HaeIIIfollowed by the addition of biotinylated dideoxynucleotides (BoehringerMannheim, Indianapolis, Ind.) to the cut ends using terminal transferase(Promega). Lanes 1, 3 and 5 contain the reaction products from theincubation of the wild type 157 nucleotide substrate in the absence ofthe DNAPTaq enzyme (lane 1), in the presence of MgCl₂ and enzyme (lane3) or in the presence of MnCl₂ and enzyme (lane 5). Lanes 2, 4 and 6contains the reaction products from the incubation of the 157 nucleotidesubstrate derived from the 419 mutant in the absence of enzyme (lane 2),in the presence of MgCl₂ and enzyme (lane 4) or in the presence of MnCl₂and enzyme (lane 6).

FIG. 30 demonstrates that the use of MnCl₂ rather than MgCl₂ in thecleavage reaction results in the production of an enhanced cleavagepattern. It is desirable that the cleavage products are of differentsizes so that the products do not all cluster at one end of the gel. Theability to spread the cleavage products out over the entire length ofthe gel makes it more likely that alterations in cleavage productsbetween the wild type and mutant substrates will be identified. FIG. 30shows that when Mg²⁺ is used as the divalent cation, the majority of thecleavage products cluster together in the upper portion of the gel. Incontrast when Mn²⁺ is used as the divalent cation, the substrate assumesstructures which, when cleaved, generate products of widely differingmobilities. These results show that Mn²⁺ is the preferred divalentcation for the cleavage reaction.

B. 5′ Nuclease Cleavage of Different but Similarly Sized DNAs GeneratesUnique Cleavage Fragments

The ability of 5′ nuclease to generate a cleavage pattern or“fingerprint” which is unique to a given piece of DNA was shown byincubating four similarly sized DNA substrates with the enzyme CLEAVASEBN. The four DNA substrates used were a 157 nucleotide fragment from thesense (or coding) strand of exon 4 of the wild-type tyrosinase gene (SEQID NO:47); a 157 nucleotide fragment from the anti-sense (or non-coding)strand of exon 4 of the wild-type tyrosinase gene (SEQ ID NO:48); a 165nucleotide DNA fragment derived from pGEM3Zf(+) (SEQ ID NO:49) and a 206nucleotide DNA fragment derived from the bottom strand of pGEM3Zf(+)(SEQ ID NO:50). The DNA substrates contained either a biotin orfluorescein label at their 5′ or 3′ ends. The substrates were made asfollows.

To produce the sense and anti-sense single-stranded substratescorresponding to exon 4 of the wild-type tyrosinase gene, adouble-stranded DNA fragment, 157 nucleotides in length (SEQ ID NO:40),was generated using symmetric PCR. The target for the symmetric PCR wasgenomic DNA containing the wild-type tyrosinase gene. The symmetric PCRcomprised 50-100 ng of genomic wild-type DNA, 25 pmoles each of primersSEQ ID NOS:42 and 43, 50 μM each dNTP and 1.25 units of Taq polymerasein 50 μl of 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05%TWEEN 20 and 0.05% NONIDET P-40. The reaction mixture was overlaid withtwo drops of light mineral oil and the tube was heated to 94° C. for 30sec, cooled to 50° C. for 1 min, heated to 72° C. for 2 min for 30repetitions. The double-stranded PCR product was gel purified,precipitated and resuspended in 40 μl of TE buffer as described above ina).

The single-stranded sense and anti-sense 157 nucleotide DNA fragmentswere generated using the above 157 by wild-type DNA fragment (SEQ IDNO:40) in two asymmetric PCR reactions. The sense strand fragment wasgenerated using 5 μl of the above purified 157 by fragment (SEQ IDNO:40) as the target in an asymmetric PCR. The reaction mixtures for theasymmetric PCR were as above for the symmetric PCR with the exceptionthat 100 pmoles of the biotin-labeled sense primer (SEQ ID NO:42) and 1pmole of the fluorescein-labeled anti-sense primer (SEQ ID NO:43) wasused to prime the reaction. The anti-sense fragment was generated using5 μl of the above purified 157 by fragment as the target in anasymmetric PCR. The reaction conditions for the asymmetric PCR were asabove for the symmetric PCR with the exception that 1 pmole of the senseprimer (SEQ ID NO:42) and 100 pmoles of the anti-sense primer (SEQ IDNO:43) was used to prime the reaction.

The reaction conditions for the asymmetric PCR were 95° C. for 45 sec,50° C. for 45 sec, 72° C. for 1 min and 15 sec for 30 repetitions with a5 min incubation at 72° C. after the last repetition. The reactionproducts were visualized, extracted and collected as described abovewith the single stranded DNA being identified by a shift in mobilitywhen compared to a double stranded DNA control.

The single-stranded 165 nucleotide fragment from pGEM3Zf(+) (SEQ IDNO:49) was generated by asymmetric PCR. The PCR comprised 50 pmoles of5′ biotin-AGCGGATAACAATTTCACACAGGA-3′ (SEQ ID NO:51; Promega) and 1pmole of 5′-CACGGATCCTAATACGACTCACTATAGGG-3′ (SEQ ID NO:52; IntegratedDNA Technologies, Coralville, Iowa), 50 μM each dNTP, 20 mM Tris-Cl, pH8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05% NONIDETP-40. Forty-five microliters of this reaction mixture was overlaid withtwo drops of light mineral oil and the tube was heated to 95° C. for 5sec and then cooled to 70° C. Taq polymerase was then added at 1.25units in 5 μl of 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with0.05% TWEEN 20 and 0.05% NONIDET P-40. The tubes were heated to 95° C.for 45 sec, cooled to 50° C. for 45 sec, heated to 72° C. for 1 min 15sec for 30 repetitions with a 5 min incubation at 72° C. after the lastrepetition. The reaction products were visualized, extracted andcollected as described above with the 164 nucleotide DNA fragment beingidentified by a shift in mobility when compared to a double stranded DNAcontrol.

The 206 nucleotide DNA fragment (SEQ ID NO:50) was prepared byasymmetric as follows. The asymmetric PCR comprised 1 pmole of adouble-stranded 206 by PCR product (generated as described in Example1C), 50 pmoles of the primer 5′-CGCCAGGGTTTTCCCAGTCACGAC-3′ (SEQ IDNO:53), 50 μM each dNTP, 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl,with 0.05% TWEEN 20 and 0.05% NONIDET P-40. Ninety-five microliters ofthis reaction mixture was overlaid with three drops of light mineral oiland the tube was heated to 95° C. for 5 sec and then cooled to 70° C.Taq polymerase was then added at 2.5 units in 5 μl of 20 mM Tris-Cl, pH8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05% NONIDETP-40. The tubes were heated to 95° C. for 45 sec, cooled to 63° C. for45 sec, heated to 72° C. for 1 min 15 sec for 15 repetitions with a 5min incubation at 72° C. after the last repetition. The reactionproducts were visualized, extracted and collected as described abovewith the 206 nucleotide DNA fragment being identified by a shift inmobility when compared to a double stranded DNA control. Theprecipitated DNA was resuspended in 70 μl of TE buffer.

Twenty-five microliters of the above product was biotinylated on the 3′end using 10-20 units of terminal deoxynucleotidyl transferase (Promega)in a 50 μl reaction. The reaction comprised 0.5 nmoles ofbiotin-16-ddUTP (Boehringer Mannheim) and 1× buffer (500 mM cacoodylatebuffer, pH 6.8, 5 mM CoCl₂, 0.5 mM DTT and 500 μg/ml BSA). The tubeswere incubated at 37° C. for 15 min followed by ethanol precipitation inthe presence of 4 μg of glyc7ogen. The DNA was ethanol precipitated asecond time and then resuspended in 25 μl of 10 Mm Tris-HCl, pH 8.0, 0.1mM EDTA.

The cleavage reactions were carried out in a final volume of 10 μlcontaining 1×CFLP buffer (10 mM MOPS, pH 8.2) with 1 mM MnCl₂ usingapproximately 100 fmoles of substrate DNA and 250 ng of the enzymeCLEAVASE BN. Parallel reactions lacking the enzyme CLEAVASE BN (noenzyme control) were set up as above with the exception that one thirdas much DNA template was used (approximately 33 fmoles of each template)to balance the signal on the autoradiograph.

Each substrate DNA was placed in a 200 μl thin wall microcentrifuge tube(BioRad, Hercules, Calif.) in 5 μl of 1×CFLP buffer with 2 mM MnCl₂. Thesolution was overlaid with one drop of light mineral oil. Tubes werebrought to 95° C. for 5 seconds to denature the substrates and then thetubes were quickly cooled to 65° C.

Cleavage reactions were started immediately by the addition of a dilutedenzyme mixture comprising 1 μl of the enzyme CLEAVASE BN (250 ng/μl in1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50mM KCl, 10 μg/ml BSA)) in 5 μl of 1×CFLP buffer without MnCl₂. Theenzyme solution was at room temperature before addition to the cleavagereaction. After 5 minutes at 65° C., the reactions were stopped by theaddition of 8 μl of stop buffer. Samples were heated to 72° C. for 2minutes and 5 μl of each reaction were resolved by electrophoresisthrough a 10% polyacrylamide gel (19:1 cross-link), with 7 M urea, in abuffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with a0.45 μm-pore positively charged nylon membrane (United StatesBiochemical). The DNA was transferred to the membrane and the membranewas dried, washed in 1.2× SEQUENASE IMAGES Blocking Buffer, treated with1×SAAP buffer as described above. The signal was developed usingLUMIPHOS-530 (United States Biochemical) or Quantum YieldChemiluminescent Substrate (Promega) in place of the CDPSTAR reagent;the membrane was then exposed to X-ray film as described above. Theresulting autoradiograph is shown in FIG. 31.

FIG. 31 shows the results of incubation of the four substrates describedabove in the presence or absence of the enzyme CLEAVASE BN. Four sets ofreactions are shown. Set one contains the reaction products from theincubation of the 157 nucleotide sense strand fragment of the tyrosinasegene (SEQ ID NO:47) in the absence or presence of the enzyme CLEAVASEBN. Set two contains the reaction products from the incubation of the157 nucleotide anti-sense strand fragment of the tyrosinase gene (SEQ IDNO:48) in the absence or presence of the enzyme CLEAVASE BN. Set threecontains the reaction products from the incubation of the 165 basebottom strand fragment of the plasmid pGEM3Zf(+) (SEQ ID NO:49) in theabsence or presence of the enzyme CLEAVASE BN. Set four contains thereaction products from the incubation of the 206 base top strandfragment of the plasmid pGEM3Zf(+) (SEQ ID NO:50) in the absence orpresence of the enzyme CLEAVASE BN. Lanes marked “M” containbiotin-labeled molecular weight markers prepared as described above; thesizes of the marker fragments are indicated in FIG. 31. In the absenceof the enzyme CLEAVASE BN, no cleavage of the substrates is observed. Inthe presence of the enzyme CLEAVASE BN, each substrate is cleavedgenerating a unique set of cleavage products. When these cleavageproducts are resolved on a polyacrylamide gel, a unique pattern orfingerprint is seen for each substrate DNA. Thus, although the foursubstrates are similar in size (157 to 206 bases), the enzyme CLEAVASEBN generates a unique collection of cleavage products from eachsubstrate. These unique cleavage patterns result from the characteristicconformation each substrate DNA assumes.

The present invention contemplates the ability to generate a uniquecleavage pattern for two or more DNA substrates of the same size as partof a method for the detection of genetic mutations. This method comparesa normal (or wild type or non-mutated) substrate with a substrate from apatient suspected of having a mutation in that substrate. The twosubstrates would be of the same length and the cleavage reaction wouldbe used to probe the patient DNA substrate for conformational changesrelative to the pattern seen in the wild type control substrate.

Example 11 Cleavage Directed by the Enzyme CLEAVASE BN Can Detect SingleBase Changes in DNA Substrates

The ability of the enzyme CLEAVASE BN to cleave DNA substrates of thesame size but which contain single base changes between the substratesis herein demonstrated. The human tyrosinase gene was chosen as a modelsystem because numerous single point mutations have been identified inexon 4 of this gene (Spritz, R. A. (1994) Human Molecular Genetics3:1469). Mutation of the tyrosinase gene leads to oculocutaneousalbinism in humans.

Three single-stranded substrate DNAs were prepared; the substratescontain a biotin label at their 5′ end. The wild type substratecomprises the 157 nucleotide fragment from the sense strand of the humantyrosinase gene ((SEQ ID NO:47); Geibel, L. B., et al. (1991) Genomics9:435). Two mutation-containing substrates were used. The 419 substrate(SEQ ID NO:54) is derived from the tyrosinase mutant G419R whichcontains a glycine (GGA) to arginine (AGA) substitution; this mutantdiffers from the wild-type exon 4 fragment by a single base change atnucleotide 2675 (King, R. A., et al. (1991) Mol. Biol. Med. 8:19). The422 substrate (SEQ ID NO:55) is derived from the tyrosinase mutant R422Qwhich contains an arginine (CGG) to glutamine (CAG) substitution; thismutant differs from the wild type exon 4 fragment by a single basechange at nucleotide 2685 (Giebel, L. B., et al. (1991) J. Clin. Invest.87:1119).

Single-stranded DNA containing a biotin label at the 5′ end wasgenerated for each substrate using asymmetric PCR as described inExample 10a with the exception that the single-stranded PCR productswere recovered from the gel rather than the double-stranded products.

The following primer pair was used to amplify each DNA (the 419 and 422mutations are located internally to the exon 4 fragment amplified by theprimer pair thus the same primer pair can be used to amplify the wildtype and two mutant templates). The primer listed as SEQ ID NO:42 (senseprimer) contains a biotin label at the 5′ end and was used in a 100-foldexcess over the anti-sense primer of SEQ ID NO:43.

To generate the single stranded substrates the following templates wereused. Ten ng of supercoiled plasmid DNA was used as the target togenerate the wild-type (plasmid pcTYR-N1Tyr) or 422 mutant (plasmidpcTYR-A422) 157 nucleotide fragments. Five microliters of the gelpurified 339 by PCR fragment (SEQ ID NO:44) derived from genomic DNAhomozygous for the 419 mutation (described in Example 10a) was used asthe target to generate the 157 nucleotide 419 mutant fragment (SEQ IDNO:54).

For each target DNA, the asymmetric PCR comprised 100 pmoles of SEQ IDNO:42 and 1 pmole of SEQ ID NO:43, 50 μM each dNTP, 20 mM Tris-Cl, pH8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05% NONIDETP-40. The reaction mixture (45 μl) was overlaid with two drops of lightmineral oil and the tubes were heated to 95° C. for 5 sec then cooled to70° C. Taq polymerase was then added as 1.25 units of enzyme in 5 μl of20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and0.05% NONIDET P-40. The tubes were heated to 95° C. for 45 sec, cooledto 50° C. for 45 sec, heated to 72° C. for 1 min 15 sec for 30repetitions with a 5 min incubation at 72° C. after the last repetition.The single stranded PCR products were gel purified, precipitated andresuspended in 40 μl of TE buffer as described above.

Cleavage reactions were performed as follows. Each substrate DNA (100fmoles) was placed in a 200 μl thin wall microcentrifuge tube (BioRad)in 5 μl of 1×CFLP buffer with 2 mM MnCl₂. A tube containing 33 fmoles oftemplate DNA in 10 μl of 1×CFLP buffer and 1 MnCl₂ was prepared for eachtemplate and served as the no enzyme (or uncut) control. The solutionwas overlaid with one drop of light mineral oil. Tubes were brought to95° C. for 5 seconds to denature the substrates and then the tubes werequickly cooled to 65° C.

Cleavage reactions were started immediately by the addition of a dilutedenzyme mixture comprising 1 μl of the enzyme CLEAVASE BN (250 ng/μl in1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50mM KCl, 10 μg/ml BSA)) in 5 μl of 1×CFLP buffer without MnCl₂. Theenzyme solution was at room temperature before addition to the cleavagereaction. After 5 minutes at 65° C., the reactions were stopped by theaddition of 8 μl of stop buffer. The samples were heated to 72° C. for 2minutes and 7 μl of each reaction were resolved by electrophoresisthrough a 10% polyacrylamide gel (19:1 cross-link), with 7 M urea, in abuffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10a. The DNA was transferred tothe membrane and the membrane was dried, washed in 1.2× SEQUENASE IMAGESBlocking Buffer, treated with 1×SAAP buffer and reacted with CDPSTARreagent (Tropix) and exposed to X-ray film as described in Example 10a.The resulting autoradiograph is shown in FIG. 32.

In FIG. 32, lanes marked “M” contain molecular weight markers preparedas described in Example 10. Lanes 1-3 contain the no enzyme control forthe wild type (SEQ ID NO:47), the 419 mutant (SEQ ID NO:54) and the 422mutant (SEQ ID NO:55) substrates, respectively. Lane 4 contains thecleavage products from the wild type template. Lane 5 contains thecleavage products from the 419 mutant. Lane 6 contains the cleavageproducts from the 422 mutant.

FIG. 32 shows that a similar, but distinctly different, pattern ofcleavage products is generated by digestion of the three template DNAswith the enzyme CLEAVASE BN. Note that in the digest of mutant 419, thebands below about 40 nucleotides are absent, when compared to wild-type,while in the digest of mutant 422 several new bands appear in the 53nucleotide range.

Although the three template DNAs differed in only one of the 157nucleotides, a unique pattern of cleavage fragments was generated foreach. Thus a single base change in a 157 nucleotide fragment gives riseto different secondary structures which are recognized by the enzymeCLEAVASE.

Example 12 Single Base Changes in Large DNA Fragments are Detected bythe Enzyme CLEAVASE BN

The previous example demonstrated that the 5′ nuclease activity of theenzyme CLEAVASE BN could be used to detect single point mutations withina 157 nucleotide DNA fragment. The ability of the enzyme CLEAVASE BN todetect single point mutations within larger DNA fragments is hereindemonstrated.

Increasingly larger fragments derived from the 422 tyrosinase mutant wascompared to the same size fragments derived from the wild-typetyrosinase gene. Four sets of single-stranded substrates wereutilized: 1) a 157 nucleotide template derived from the sense strand ofexon 4 from the wild-type (SEQ ID NO:47) and 422 mutant (SEQ ID NO:55),2) a 378 nucleotide fragment containing exons 4 and 5 from the wild-type(SEQ ID NO:56) and 422 mutant (SEQ ID NO:57), 3) a 1.059 kb fragmentcontaining exons 1-4 from the wild-type (SEQ ID NO:58) and 422 mutant(SEQ ID NO:59) and 4) a 1.587 kb fragment containing exons 1-5 from thewild-type (SEQ ID NO:60) and 422 mutant (SEQ ID NO:61). The onlydifference between the wild type and 422 mutant templates is the G to Achange in exon 4 regardless of the length of the template used. The G toA point mutation is located 27, 27, 929 and 1237 nucleotides from thelabeled ends of the 157 base, 378 base, 1.059 kb and 1.6 kb substrateDNAs, respectively.

a) Preparation of the Substrate DNA

A cDNA clone containing either the wild-type (pcTYR-N1Tyr, Bouchard, B.,et al. (1989) J. Exp. Med. 169:2029) or 422 mutant (pcTYR-A422, Giebel,L. B., et al. (1991) 87:1119) tyrosinase gene was utilized as the targetDNA in PCRs to generate the above substrate DNAs. The primer pairconsisting of SEQ ID NOS:42 and 43 were used to generate a doublestranded 157 by DNA fragment from either the mutant of wild-type cDNAclone. The primer pair consisting of SEQ ID NO:42 and SEQ ID NO:62 wasused to generate a double stranded 378 by DNA fragment from either thewild-type or mutant cDNA clone. The primer pair consisting of SEQ IDNO:63 and SEQ ID NO:43 was used to generate a double stranded 1.059 kbpDNA fragment from either the wild-type or mutant cDNA clone. The primerpair consisting of SEQ ID NO:64 and SEQ ID NO:62 was used to generate adouble stranded 1.587 kbp DNA fragment from either the wild-type ormutant cDNA clone. In each case the sense strand primer contained abiotin label at the 5′ end.

The PCR reactions were carried out as follows. One to two ng of plasmidDNA from the wild-type or 422 mutant was used as the target DNA in a 100μl reaction containing 50 μM of each dNTP, 1 μM of each primer in agiven primer pair, 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with0.05% TWEEN 20 and 0.05% NONIDET P-40. Tubes containing the abovemixture were overlaid with three drops of light mineral oil and thetubes were heated to 94° C. for 1 min, then cooled to 70° C. Taqpolymerase was then added as 2.5 units of enzyme in 5 μl of 20 mMTris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05%NONIDET P-40. The tube was heated to 93° C. for 45 sec, cooled to 52° C.for 2 min, heated to 72° C. for 1 min 45 sec for 35 repetitions, with a5 min incubation at 72° C. after the last repetition.

Following the PCR, excess primers were removed using a QIA QUICK-SPINPCR Purification kit (Qiagen, Inc. Chatsworth, Calif.) following themanufacturer's instructions; the DNA was eluted in 50 μl of 10 mMTris-HCl, pH 8.0, 1 mM EDTA. The sense strand of each of thedouble-stranded fragments from the wild-type and 422 mutant gene wereisolated as follows. Streptavidin-coated paramagnetic beads (Dynal M280beads) (0.5 mg in 50 μl; pre-washed in 2× bind and wash (B&W) buffer (2M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% TWEEN 20)) were added toeach purified PCR product. The samples were incubated at roomtemperature for 15 minutes with occasional shaking. The beads wereremoved from the supernatant by exposing the tube to a magnetic plateand the supernatant was discarded. The bead-DNA complexes were washedtwice in 2× B&W buffer. One hundred microliters of 0.1 M NaOH were addedto the beads and the samples were incubated at room temperature for 15minutes (for the 157, 378 by DNAs); for DNA fragments larger than 1 kb,the beads were incubated at 47° C. for 30 minutes. After incubation, thebeads were washed twice with 2× B&W buffer. Finally, the bead-ssDNAcomplexes were resuspended in 50 μl 2× B&W buffer and stored at 4° C.

b) Cleavage Reaction Conditions

The cleavage reactions were performed directly on the single-strandedDNA-bead complexes. Five to 10 μl of DNA-bead complex (about 100 fmolesof DNA) were placed in a 200 μl microcentrifuge tube and washed oncewith 10 μl of sterile H₂O, Seven and one half microliters of 1×CFLPbuffer with 1.3 mM MnCl₂ (to yield a final concentration of 1 mM) wasthen added to each tube. The reaction tubes were prewarmed to 65° C. for2 minutes and cleavage was initiated by the addition of 2.5 μl of theenzyme CLEAVASE BN (10-50 ng in 1× dilution buffer). The reaction wascarried out at 65° C. for 5 min.

Immediately after this 5 min incubation, the beads were allowed tosettle to the bottom of the tube and the supernatant was removed anddiscarded. Ten to forty microliters of stop buffer (95% formamide with20 mM EDTA and 0.05% xylene cyanol and 0.05% bromophenol blue) was thenadded to the beads and the sample was incubated at 90° C. for 5-10minutes. The formamide/EDTA solution releases the biotinylated DNA fromthe beads. The beads were allowed to settle to the bottom of the tube.The supernatant containing the cleavage products was collected. Two toeight microliters of the supernatant solution loaded onto 6%polyacrylamide gel (19:1 cross-link), with 7 M urea, in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10a and allowed to transferovernight. After transfer the membrane was dried, blocked, probed andwashed as described in Example 10a. The blot was reacted with CDPSTARreagent (Tropix) and exposed to X-ray film as described in Example 10a.The resulting autoradiograph is shown in FIG. 33.

In FIG. 33, lanes marked “M” contain molecular weight markers preparedas described in Example 10. Lanes 1, 3, 5 and 7 contain cleavageproducts using the 157, 378, 1056 or 1587 nucleotide sense strandfragment from the wild-type tyrosinase gene, respectively. Lanes 2, 4, 6and 8 contain cleavage products using the 157, 378, 1056 or 1587nucleotide sense strand fragment from the 422 mutant tyrosinase gene,respectively.

As shown in FIG. 33, the clear pattern of cleavages seen between thewild type and 422 mutant was not obscured when the single base changewas located in longer DNA fragments. Thus, the cleavage reaction of theinvention can be used to scan large fragments of DNA for mutations.Fragments greater than about 500 by in length cannot be scanned usingexisting methodologies such as SSCP or DGGE analysis.

Example 13 The CLEAVASE Enzyme Reaction is Insensitive to Large Changesin Reaction Conditions

The results shown above demonstrated that the enzyme CLEAVASE BN can beused to probe DNA templates in a structure-specific but sequenceindependent manner. These results demonstrated that the enzyme CLEAVASEBN could be used as an efficient way to recognize conformational changesin nucleic acids caused by sequence variations. This suggested that the5′ nuclease activity of the enzyme CLEAVASE BN could be used to developa method to scan nucleic acid templates for sequence alterationsrelative to a wild-type template. The experiments below showed that thiswas the case. Furthermore it is demonstrated below that the method ofthe invention is relatively insensitive to large changes in conditionsthereby making the method suitable for practice in clinicallaboratories.

First, the effect of varying the concentration of MnCl₂ on the cleavagereaction was determined. Second, the effect of different amounts of salt(KCl) on the cleavage pattern was examined. Third, a time course wasperformed to investigate when complete cleavage was obtained. Fourth, atemperature titration was performed to determine the effect oftemperature variations on the cleavage pattern. Next, the enzyme wastitrated to determine the effect of a 50-fold variation in enzymeconcentration on the cleavage reaction. The results of these experimentsshowed that the CLEAVASE enzyme reaction is remarkably robust to largechanges in conditions.

a) MnCl₂ Titration

To determine the sensitivity of the cleavage reaction to fluctuations inthe concentration of MnCl₂, a single template was incubated in thepresence of a fixed amount of the enzyme CLEAVASE BN (250 ng) in abuffer containing 10 mM MOPS, pH 8.2 and various amount of MnCl₂. Thecleavage reaction was performed as follows. One hundred fmoles of the157 nucleotide sense strand fragment of the tyrosinase gene (SEQ IDNO:55; prepared by asymmetric PCR as described in Example 11) was placedin a 200 μl thin wall microcentrifuge tube (BioRad) in 5 μl of 1×CFLPbuffer with 0, 2, 4, 8, 12 or 20 mM MnCl₂ (to yield a finalconcentration of either 0, 1, 2, 4, 6, 8 or 10 mM MnCl₂). A tubecontaining 100 fmoles template DNA in 5 μl of 1×CFLP buffer with 10MnCl₂ was prepared and served as the no enzyme (or uncut) control. Eachreaction mixture was overlaid with a drop of light mineral oil. Thetubes were heated to 95° C. for 5 sec and then cooled to 65° C.

Cleavage reactions were started immediately by the addition of a dilutedenzyme mixture comprising 1 μl of the enzyme CLEAVASE BN (250 ng/μl in1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50mM KCl, 10 μg/ml BSA)) in 5 μl of 1×CFLP buffer without MnCl₂. Theenzyme solution was at room temperature before addition to the cleavagereaction. After 5 minutes at 65° C., the reactions were stopped by theaddition of 8 μl of stop buffer. Samples were heated to 72° C. for 2minutes and 8 μl of each reaction were resolved by electrophoresisthrough a 10% polyacrylamide gel (19:1 cross-link), with 7 M urea, in abuffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1.2× SEQUENASE IMAGESBlocking Buffer, treated with 1×SAAP buffer and reacted withLUMIPHOS-530 (United States Biochemical) or Quantum YieldChemiluminescent Substrate (Promega Corp., Madison Wis.) and exposed toX-ray film as described in Example 10. The resulting autoradiograph isshown in FIG. 34.

In FIG. 34, lanes marked “M” contain molecular weight markers. Lane 1contains the no enzyme control and shows the migration of the uncleavedtemplate DNA. Lanes 2 through 8 contain reaction products incubated inthe presence of the enzyme CLEAVASE BN in a buffer containing 10, 8, 6,4, 2, 1, or 0 mM MnCl₂, respectively.

FIG. 34 shows that no cleavage occurs in the absence of divalent cations(lane 8, 0 mM MnCl₂). Efficient production of cleavage fragments waspromoted by the inclusion of MnCl₂. The most distinct pattern ofcleavage seen at 1 mM MnCl₂ (lane 7), but little change in the patternwas seen when the Mn²⁺ concentration varied from 1 to 4 mM; Highconcentrations of MnCl₂ tend to suppress the cleavage reaction(concentrations above 6 mM). These results show that the cleavagereaction requires a divalent cation but that changes in the amount ofdivalent cation present have little effect upon the cleavage pattern.

b) Effect of Salt Concentration on the Cleavage Reaction

To determine the effect of salt concentration upon the cleavagereaction, a single template was incubated in the presence of a fixedamount of the enzyme CLEAVASE BN (250 ng) in a buffer containing 10 mMMOPS, pH 8.2, 1 mM MnCl₂ and various amount of KCl.

One hundred fmoles of the 157 base fragment derived from the sensestrand of exon 4 of the tyrosinase gene (SEQ ID NO:47; prepared asdescribed in Example 10a) was placed in a 200 μl thin wallmicrocentrifuge tube (BioRad) in a buffer containing 10 mM MOPS, pH 8.2and 1 mM MnCl₂. KCl was added to give a final concentration of either 0,10, 20, 30, 40, or 50 mM KCl; the final reaction volume was 10 μl.

A tube containing 10 mM MOPS, pH 8.2, 1 mM MnCl₂, 33 fmoles template DNAand 50 mM KCl was prepared and served as the no enzyme (or uncut)control. Each reaction mixture was overlaid with a drop of light mineraloil. The tubes were heated to 95° C. for 5 seconds and then cooled to65° C.

Cleavage reactions were started immediately by the addition of a dilutedenzyme mixture comprising 1 μl of the enzyme CLEAVASE BN (250 ng/μl in1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50mM KCl, 10 μg/ml BSA)) in 5 μl of 1×CFLP buffer without MnCl₂. Theenzyme solution was at room temperature before addition to the cleavagereaction. After 5 minutes at 65° C., the reactions were stopped by theaddition of 8 μl of stop buffer. Samples were heated to 72° C. for 2minutes and 8 μl of each reaction were resolved by electrophoresisthrough a 10% polyacrylamide gel (19:1 cross-link), with 7 M urea, in abuffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1.2× SEQUENASE IMAGESBlocking Buffer, treated with 1×SAAP buffer and reacted withLUMIPHOS-530 (United States Biochemical) or Quantum YieldChemiluminescent Substrate (Promega Corp., Madison Wis.) and exposed toX-ray film as described in Example 10. The resulting autoradiograph isshown in FIG. 35.

In FIG. 35, lanes marked “M” contain molecular weight markers. Lane 1contains the no enzyme control and shows the migration of the uncleavedtemplate DNA. Lanes 2 through 7 contain reaction products incubated inthe presence of the enzyme CLEAVASE BN in a buffer containing 50, 40,30, 20, 10 or 0 mM KCl, respectively.

The results shown in FIG. 35 show that the CLEAVASE enzyme reaction isrelatively insensitive to variations in salt concentration. The samecleavage pattern was obtained when the 157 nucleotide tyrosinase DNAtemplate (SEQ ID NO:47) was incubated with the enzyme CLEAVASEregardless of whether the KCl concentration varied from 0 to 50 mM.

c) Time Course of the Cleavage Reaction

To determine how quickly the cleavage reaction is completed, a singletemplate was incubated in the presence of a fixed amount of the enzymeCLEAVASE BN for various lengths of time. A master mix comprising 20 μlof a solution containing 1×CFLP buffer, 2 mM MnCl₂, and 400 fmoles ofthe 157 base fragment derived from the sense strand of exon 4 of thetyrosinase gene ((SEQ ID NO:47); prepared as described in Example 10b)was made. Five microliter aliquots were placed in 200 μl thin wallmicrocentrifuge tube (BioRad) for each time point examined. A no enzymecontrol tube was run; this reaction contained 33 fmoles of the templateDNA in 1×CFLP buffer with 1 mM MnCl₂ (in a final reaction volume of 10μl). The solutions were overlaid with one drop of light mineral oil. Thetubes were brought to 95° C. for 5 seconds to denature the templates andthen the tubes were cooled to 65° C.

Cleavage reactions were started immediately by the addition of a dilutedenzyme mixture comprising 1 μl of the enzyme CLEAVASE BN (250 ng per μlin 1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0,50 mM KCl, 10 μg/ml BSA)) in 5 μl of 1×CFLP buffer without MnCl₂.Immediately at the indicated time points, the reaction was stopped bythe addition of 8 μl of 95% formamide containing 20 mM EDTA and 0.05%each xylene cyanol and bromophenol blue. The no enzyme control wasincubated at 65° C., for 10 minutes and treated in the same manner asthe other reactions by the addition of 8 μl of stop buffer. Samples wereheated to 72° C. for 2 minutes and 5 μl of each reaction were resolvedby electrophoresis through a 10% polyacrylamide gel (19:1 cross-link),with 7 M urea, in a buffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mMEDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1.2× SEQUENASE IMAGESBlocking Buffer, treated with 1×SAAP buffer and reacted withLUMIPHOS-530 (United States Biochemical) or Quantum YieldChemiluminescent Substrate (Promega Corp., Madison Wis.) and exposed toX-ray film as described in Example 10b. The resulting autoradiograph isshown in FIG. 36.

In FIG. 36, lanes marked “M” contain molecular weight markers preparedas described in Example 10. Lane 1 contains the no enzyme controlincubated for 10 minutes. Lanes 2-5 contain the cleavage products fromreactions incubated for 0.1, 1, 5 or 10 minutes at 65° C. FIG. 36 showsthat the cleavage reaction mediated by the enzyme CLEAVASE BN is veryrapid. Cleavage is already apparent at less than 6 seconds (<0.1 min)and is complete within one minute. These results also show that the samepattern of cleavage is produced whether the reaction is run for 1 or 10minutes.

d) Temperature Titration of the CLEAVASE Enzyme Reaction

To determine the effect of temperature variation on the cleavagepattern, the 157 base fragment derived from the sense strand of exon 4of the tyrosinase gene (SEQ ID NO:47) was incubated in the presence of afixed amount of the enzyme CLEAVASE BN for 5 minutes at varioustemperatures. One hundred fmoles of substrate DNA (prepared as describedin Example 10b) was placed in a 200 μl thin wall microcentrifuge tube(BioRad) in 5 μl of 1×CFLP buffer with 2 mM MnCl₂. Two “no enzyme” testcontrol tubes were set-up as above with the exception that thesereactions contained 33 fmoles of substrate DNA in 10 μl of the abovebuffer with 1 mM MnCl₂. The solution was overlaid with one drop of lightmineral oil. Tubes were brought to 95° C. for 5 seconds to denature thetemplates and then the tubes were cooled to the desired temperature.

Cleavage reactions were started immediately by the addition of a dilutedenzyme mixture comprising 1 μl of the enzyme CLEAVASE BN (250 ng per μlin 1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0,50 mM KCl, 10 μg/ml BSA)) in 5 μl of 1×CFLP buffer without MnCl₂. Thetubes placed at either 55°, 60°, 65°, 70°, 75° or 80° C. After 5 minutesat a given temperature, the reactions were stopped by the addition of 8μl of stop buffer.

Samples were heated to 72° C. for 2 minutes and 5 μl of each reactionwere resolved by electrophoresis through a 10% polyacrylamide gel (19:1cross-link), with 7 M urea, in a buffer containing 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1.2× SEQUENASE IMAGESBlocking Buffer, treated with 1×SAAP buffer and reacted withLumiphos-530 (United States Biochemical) or Quantum YieldChemiluminescent Substrate (Promega Corp., Madison Wis.) and exposed toX-ray film as described in Example 10. The resulting autoradiograph isshown in FIG. 37.

In FIG. 37, the lanes marker “M” contain molecular weight markersprepared as described in Example 10. Lanes 1 and 2 contain no enzymecontrols incubated at 55° C. and 80° C., respectively. Lanes 3-8 containthe cleavage products from the enzyme CLEAVASE-containing reactionsincubated at 55° C., 60° C., 65° C., 70° C., 75° C. or 80° C.,respectively.

FIG. 37 shows that the CLEAVASE enzyme reaction can be performed over awide range of temperatures. The pattern of cleavages changedprogressively in response to the temperature of incubation, in the rangeof 55° C. to 75° C. Some bands were evident only upon incubation athigher temperatures. Presumably some structures responsible for cleavageat the intermediate temperatures were not favored at the lowertemperatures. As expected, cleavages became progressively less abundantin the high end of the temperature range tested as structures weremelted out. At 80° C. cleavage was inhibited completely presumably dueto complete denaturation of the template.

These results show that the cleavage reaction can be performed over awide range of temperatures. The ability to run the cleavage reaction atelevated temperatures is important. If a strong (i.e., stable) secondarystructure is assumed by the templates, a single nucleotide change isunlikely to significantly alter that structure, or the cleavage patternit produces. Elevated temperatures can be used to bring structures tothe brink of instability, so that the effects of small changes insequence are maximized, and revealed as alterations in the cleavagepattern within the target template, thus allowing the cleavage reactionto occur at that point.

e) Titration of the Enzyme CLEAVASE BN

The effect of varying the concentration of the enzyme CLEAVASE BN in thecleavage reaction was examined. One hundred fmoles of the 157 basefragment derived from the sense strand of exon 4 of the tyrosinase gene(SEQ ID NO:47) was placed in 4 microcentrifuge tubes in 5 μl of 1×CFLPbuffer with 2 mM MnCl₂. A no enzyme control tube was run; this reactioncontained 33 fmoles of substrate DNA in 10 μl of 1×CFLP buffercontaining 1 mM MnCl₂. The solutions were overlaid with one drop oflight mineral oil. The tubes were brought to 95° C. for 5 seconds todenature the templates and then the tubes were cooled to 65° C.

Cleavage reactions were started immediately by the addition of a dilutedenzyme mixture comprising 1 μl of the enzyme CLEAVASE BN in 1× dilutionbuffer such that 10, 50, 100 or 250 ng of enzyme was in the tubes in 5μl of 1×CFLP buffer without MnCl₂. After 5 minutes at 65° C., thereactions were stopped by the addition of 8 μl of stop buffer. Thesamples were heated to 72° C. for 2 minutes and 7 μl of each reactionwere resolved by electrophoresis through a 10% polyacrylamide gel (19:1cross-link), with 7 M urea, in a buffer containing 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1.2× SEQUENASE IMAGESBlocking Buffer, treated with 1×SAAP buffer and reacted withLUMIPHOS-530 (United States Biochemical) or Quantum YieldChemiluminescent Substrate (Promega Corp., Madison Wis.) and exposed toX-ray film as described in Example 10. The resulting autoradiograph isshown in FIG. 38.

The lanes marked “M” in FIG. 38 contain molecular weight markers. Lane 1contains the no enzyme control and shows the migration of the uncutsubstrate. Lanes 2-5 contain reaction products from reactions containing10, 50, 100 or 250 ng of the enzyme CLEAVASE BN, respectively.

These results show that the same cleavage pattern was obtained using the157 nucleotide tyrosinase DNA substrate regardless of whether the amountof enzyme used in the reaction varied over a 25-fold range. Thus, themethod is ideally suited for practice in clinical laboratories wherereactions conditions are not as controlled as in research laboratories.

f) Consistent Cleavage Patterns are Obtained Using Different DNAPreparations

To demonstrate that the same cleavage pattern is consistently obtainfrom a given substrate, several different preparations of the 157 basefragment derived from the sense strand of exon 4 of the tyrosinase gene(SEQ ID NO:47) were generated. The substrate was generated as describedin Example 10b. Three independent PCR reactions performed on separatedays were conducted. One of these PCR samples was split into two and onealiquot was gel-purified on the day of generation while the otheraliquot was stored at 4° C. overnight and then gel-purified the nextday.

Cleavage reactions were performed as described in Example 10b. Sampleswere run on an acrylamide gel and processed as described in Example 10b.The resulting autoradiograph is shown in FIG. 39.

In FIG. 39, the lanes marked “M” contain biotinylated molecular weightmarkers. Set 1 contains the products from a cleavage reaction performedin the absence (−) or presence (+) of enzyme on preparation no. 1. Set 2contains the products from a cleavage reaction performed in the absence(−) or presence (+) of enzyme on preparation no. 2. Set 3 contains theproducts from a cleavage reaction performed in the absence (−) orpresence (+) of enzyme on preparation no. 3. Preparation no. 3 wasderived from preparation 2 and is identical except that preparation no.3 was gel-purified one day after preparation no. 2. Set 4 contains theproducts from a cleavage reaction performed in the absence (−) orpresence (+) of enzyme on preparation no. 4. The same pattern ofcleavage products is generated from these independently preparedsubstrate samples.

These results show that independently produced preparations of the 157nucleotide DNA fragment gave identical cleavage patterns. Thus, theCLEAVASE enzyme reaction is not effected by minor differences presentbetween substrate preparations.

Example 14 Point Mutations are Detected Using Either the Sense orAnti-Sense Strand of the Tyrosinase Gene

The ability of the enzyme CLEAVASE to create a unique pattern ofcleavage products (i.e., a fingerprint) using either the sense (coding)or anti-sense (non-coding) strand of a gene fragment was examined.

Single stranded DNA substrates corresponding to either the sense (SEQ IDNO:47) or anti-sense strand (SEQ ID NO:48) of the 157 nucleotidefragment derived from the wild-type tyrosinase gene were prepared usingasymmetric PCR as described in Example 10a. The sense strand wild-typesubstrate contains a biotin label at the 5′ end; the anti-sense strandcontains a fluorescein label at the 5′ end.

A single stranded DNA substrate corresponding to the sense strand of the157 nucleotide fragment derived from the 419 mutant tyrosinase gene (SEQID NO:54) was prepared using asymmetric PCR as described in Example 11.The sense strand 419 mutant substrate contains a biotin label at the 5′end.

A single stranded DNA substrate corresponding to the anti-sense strandof the 157 nucleotide fragment derived from the 419 mutant tyrosinasegene (SEQ ID NO:65) was prepared using asymmetric PCR as described inExample 11 with the exception that 100 pmoles of the fluorescein-labeledanti-sense primer (SEQ ID NO:43) and 1 pmole of the biotin-labelledsense primer (SEQ ID NO:42) were used. The resulting anti-sense strand419 mutant substrate contains a fluorescein label at the 5′ end.

A single stranded DNA substrate corresponding to the sense strand of the157 nucleotide fragment derived from the 422 mutant tyrosinase gene (SEQID NO:55) was prepared using asymmetric PCR as described in Example 11.The sense strand 422 mutant substrate contains a biotin label at the 5′end.

A single stranded DNA substrate corresponding to the anti-sense strandof the 157 nucleotide fragment derived from the 422 mutant tyrosinasegene (SEQ ID NO:66) was prepared using asymmetric PCR as described inExample 11 with the exception that 100 pmoles of the fluorescein-labeledanti-sense primer (SEQ ID NO:43) and 1 pmole of the biotin-labelledsense primer (SEQ ID NO:42) were used. The resulting anti-sense strand422 mutant substrate contains a fluorescein label at the 5′ end.

Following asymmetric PCR, the single stranded PCR products were gelpurified, precipitated and resuspended in 40 μl of TE buffer asdescribed in Example 10.

Cleavage reactions were performed as described in Example 11. Followingthe cleavage reaction, the samples were resolved by electrophoresis asdescribed in Example 10a. After electrophoresis, the gel plates wereseparated allowing the gel to remain flat on one plate. A 0.2 μm-porepositively-charged nylon membrane (Schleicher and Schuell, Keene, N.H.),pre-wetted in 0.5×TBE (45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA), was laidon top of the exposed acrylamide gel. All air bubbles trapped betweenthe gel and the membrane were removed. Two pieces of 3MM filter paper(Whatman) were then placed on top of the membrane, the other glass platewas replaced, and the sandwich was clamped with binder clips. Transferwas allowed to proceed overnight. After transfer, the membrane wascarefully peeled from the gel and allowed to air dry. After completedrying, the membrane was washed twice in 1.5× SEQUENASE IMAGES BlockingBuffer (United States Biochemical) for 30 minutes/wash. Three tenths ofa ml of the buffer was used per cm² of membrane. The following reagentswere added directly to the blocking solution: a streptavidin-alkalinephosphatase conjugate (SAAP, United States Biochemical) added at a1:4000 final dilution and an anti-fluorescein antibody (Fab)-alkalinephosphatase conjugate (Boehringer Mannheim Biochemicals, Indianapolis,Ind.) added at a 1:20,000 final dilution. The membrane was agitated for15 minutes. The membrane was rinsed briefly with H₂O and then washed 3times (5 minutes/wash) in 1×SAAP buffer (100 mM Tris-HCL, pH 10; 50 mMNaCl) with 0.05% SDS and 0.025% TWEEN 20 using 0.5 ml buffer/cm² of thebuffer, with brief H₂O rinses between each wash. The membrane was thenwashed once in 1×SAAP buffer without SDS or TWEEN 20, drained thoroughlyand placed in a plastic heat-sealable bag. Using a sterile pipet tip,0.05 ml/cm² of CDPSTAR reagent (Tropix, Bedford, Mass.) was added to thebag and distributed over the entire membrane for 5 minutes. The bag wasdrained of all excess liquid and air bubbles. The membrane was thenexposed to X-ray film (Kodak XRP) for an initial 30 minutes. Exposuretimes were adjusted as necessary for resolution and clarity. Theresulting autoradiograph is shown in FIG. 40.

In FIG. 40, lanes marked “M” contain biotinylated molecular weightmarkers prepared as described in Example 10. Lanes 1-6 containbiotinylated sense strand substrates from the wild-type, 419 and 422mutant 157 nucleotide fragments. Lanes 1-3 contain no enzyme controlsfor the wild-type, 419 and 422 mutant fragments, respectively. Lanes 4-6contain the reaction products from the incubation of the sense strand ofthe wild-type, 419 and 422 mutant fragments with the enzyme CLEAVASE BN,respectively. Lanes 7-12 contain fluoresceinated anti-sense strandsubstrates from the wild-type, 419 and 422 mutant 157 nucleotidefragments. Lanes 1-3 contain “no enzyme” controls for the wild-type, 419and 422 mutant fragments, respectively. Lanes 4-6 contain the reactionproducts from the incubation of the anti-sense strand of the wild-type,419 and 422 mutant fragments with the enzyme CLEAVASE BN, respectively.

As expected, distinct but unique patterns of cleavage products aregenerated for the wild-type, 419 and 422 mutant fragments when eitherthe sense or anti-sense fragment is utilized. The ability to use eitherthe sense or anti-sense strand of a gene as the substrate isadvantageous because under a given set of reaction conditions one of thetwo strands may produce a more desirable banding pattern (i.e., thecleavage products are spread out over the length of the gel rather thanclustering at either end), or may have a mutation more favorably placedto create a significant structural shift. This could be more importantin the analysis of long DNA substrates which contain mutations closer toone end or the other. Additionally, detection on both strands serves asa confirmation of a sequence change.

Example 15 Detection of Mutations in the Human Beta-Globin Gene Usingthe Enzyme CLEAVASE

The results shown in Examples 10-14 showed that the CLEAVASE enzymereaction could be used to detect single base changes in fragments of thetyrosinase gene ranging from 157 nucleotides to 1.6 kb. To demonstratethat the CLEAVASE enzyme reaction is generally applicable for thedetection of mutations, a second model system was examined.

The human β-globin gene is known to be mutated in a number ofhemoglobinopathies such as sickle cell anemia and β-thalassemia. Thesedisorders generally involve small (1 to 4) nucleotide changes in the DNAsequence of the wild type β-globin gene (Orkin, S. H. and Kazazian, H.H., Jr. (1984) Annu Rev. Genet. 18:131 and Collins, F. S, and Weissman,S. M. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31:315). At least 47different mutations in the β-globin gene have been identified which giverise to a β-thalassemia.

Three β-globin mutants were compared to the wild type β-globin gene(Lawn, R. M., et al. (1980) Cell 21:647) using the CLEAVASE enzymereaction. Mutant 1 contains a nonsense mutation in codon 39; thewild-type sequence at codon 39 is CAG; the mutant 1 sequence at thiscodon is TAG (Orkin, S. H. and Goff, S. C. (1981) J. Biol. Chem.256:9782). Mutant 2 contains a T to A substitution in codon 24 whichresults in improper splicing of the primary transcript (Goldsmith, M.E., et al. (1983) Proc. Natl. Acad. Sci. USA 80:2318). Mutant 3 containsa deletion of two A residues in codon 8 which results in a shift in thereading frame; mutant 3 also contains a silent C to T substitution incodon 9 (Orkin, S. H. and Goff, S. C. (1981) J. Biol. Chem. 256:9782).

a) Preparation of Wild Type and Mutant β-Globin Gene Substrates

Single stranded substrate DNA was prepared from the above wild type andmutant β-globin genes as follows. Bacteria harboring the appropriateplasmids were streaked onto antibiotic plates and grown overnight at 37°C. (bacteria with the wild-type plasmid and the plasmid containing themutant 3, were grown on tetracycline plates; bacteria with the plasmidscontaining the mutant 1 and mutant 2 sequences were grown on ampicillinplates). Colonies from the plates were then used to isolate plasmid DNAsusing the WIZARD Minipreps DNA Purification System (Promega Corp.,Madison, Wis.). The colonies were resuspended in 200 μl of “CellResuspension Buffer” from the kit. The DNA was extracted according tothe manufacturers protocol. Final yields of approximately 2.5 μg of eachplasmid were obtained.

A 536 (wild-type, mutants 1 and 2) or 534 (mutant 3) nucleotide fragmentwas amplified from each of the above plasmids in polymerase chainreactions comprising 5 ng of plasmid DNA, 25 pmoles each of5′-biotinylated KM29 primer (SEQ ID NO:67) and 5′-fluorescein labeledRS42 primer (SEQ ID NO:68), 50 μM each dNTP and 1.25 units of Taq DNAPolymerase in 50 μl of 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KClwith 0.05% each TWEEN-20 and NONIDET P-40. The reactions were overlaidwith 2 drops of light mineral oil and were heated to 95° C. for 30seconds, cooled to 55° C. for 30 seconds, heated to 72° C. for 60seconds, for 35 repetitions in a thermocycler (MJ Research, Watertown,Mass.). The products of these reactions were purified from the residualdNTPs and primers by use of a WIZARD PCR Cleanup kit (Promega Corp.,Madison, Wis.), leaving the duplex DNA in 50 μl of 10 mM Tris-CL, pH8.0, 0.1 mM EDTA.

To generate single stranded copies of these DNAs, the PCRs describedabove were repeated using 1 μl of the duplex PCR DNA as template, andomitting the RS42 primer. The products of this asymmetric PCR wereloaded directly on a 6% polyacrylamide gel (29:1 cross-link) in a bufferof 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, alongside an aliquot of theoriginal PCR DNA to identify the location of the double-strand DNAproduct. After electrophoretic separation, the DNAs were visualized bystaining with ethidium bromide and the single stranded DNAs, identifiedby altered mobility when compared to the duplex DNAs, were excised andeluted from the gel slices by passive diffusion overnight into asolution comprising 0.5 M NH₄OAc, 0.1% SDS and 0.2 mM EDTA. The productswere collected by ethanol precipitation and dissolved in 40 μl of 10 mMTris-Cl, pH 8.0, 0.1 mM EDTA.

The sequence of the 536 nucleotide fragment from the wild-type β-globingene is listed in SEQ ID NO:69. The sequence of the 534 nucleotidefragment from mutant 3 is listed in SEQ ID NO:70. The sequence of the536 nucleotide fragment from mutant 1 is listed in SEQ ID NO:71. Thesequence of the 536 nucleotide fragment from mutant 2 is listed in SEQID NO:72.

b) Optimization of the Cleavage Reaction Using the Wild-Type Beta-GlobinSubstrate

The optimal conditions (salt concentration, temperature) which producean array of cleavage products having widely differing mobilities fromthe β-globin substrate were determined. Conditions which produce acleavage pattern having the broadest spread array with the most uniformintensity between the bands were determined (the production of such anarray of bands aids in the detection of differences seen between awild-type and mutant substrate). This experiment involved running thecleavage reaction on the wild type β-globin substrate (SEQ ID NO:69) atseveral different temperatures in the presence of either no KCl or 50 mMKCl.

For each KCL concentration to be tested, 30 μl of a master mixcontaining DNA, CFLP buffer and salts was prepared. For the “0 mM KCl”reactions, the mix included approximately 500 fmoles of single-stranded,5′ biotinylated 536-mer PCR DNA from plasmid pHBG1 in 30 μl of 1×CFLPbuffer (10 mM MOPS, pH 8.2) with 1.7 mM MnCl₂ (for 1 mM in the finalreaction); the “50 mM KCl” mix included 83.3 mM KCl in addition to theabove components. The mixes were distributed into labeled reaction tubesin 6 μl aliquots, and stored on ice until use. An enzyme dilutioncocktail included 450 ng of the enzyme CLEAVASE BN in 1×CFLP bufferwithout MnCl₂.

Cleavage reactions were performed at 60° C., 65° C., 70° C. and 75° C.For each temperature to be tested, a pair of tubes with and without KClwere brought to 95° C. for 5 seconds, then cooled to the selectedtemperature. The reactions were then started immediately by the additionof 4 μl of the enzyme cocktail. In the 75° C. test, a duplicate pair oftubes was included, and these tubes received 4 μl of 1×CFLP bufferwithout MnCl₂ in place of the enzyme addition. No oil overlay was used.All reactions proceeded for 5 minutes, and were stopped by the additionof 8 μl of stop buffer. Completed and yet-to-be-started reactions werestored on ice until all reactions had been performed. Samples wereheated to 72° C. for 2 minutes and 5 μl of each reaction was resolved byelectrophoresis through a 6% polyacrylamide gel (19:1 cross-link), with7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Afterelectrophoresis, the gel plates were separated allowing the gel toremain flat on one plate. A 0.2 μm-pore positively-charged nylonmembrane (NYTRAN, Schleicher and Schuell, Keene, N.H.), pre-wetted inH₂O, was laid on top of the exposed gel. All air bubbles were removed.Two pieces of 3MM filter paper (Whatman) were then placed on top of themembrane, the other glass plate was replaced, and the sandwich wasclamped with binder clips. Transfer was allowed to proceed overnight.After transfer, the membrane was carefully peeled from the gel andallowed to air dry. After complete drying the membrane was washed in1.2× SEQUENASE IMAGES Blocking Buffer (United States Biochemical) using0.3 ml of buffer/cm² of membrane. The wash was performed for 30 minutes.A streptavidin-alkaline phosphatase conjugate (SAAP, United StatesBiochemical) was added to a 1:4000 dilution directly to the blockingsolution, and agitated for 15 minutes. The membrane was rinsed brieflywith H₂O and then washed three times for 5 minutes per wash using 0.5ml/cm² of 1×SAAP buffer (100 mM Tris-HCl, pH 10, 50 mM NaCl) with 0.1%sodium dodecyl sulfate (SDS). The membrane was rinsed briefly with H₂Obetween each wash. The membrane was then washed once in 1×SAAP/1 mMMgCl₂ without SDS, drained thoroughly and placed in a plasticheat-sealable bag. Using a sterile pipet, 5 mls of either CSPD orCDPSTAR reagent (Tropix, Bedford, Mass.) chemiluminescent substrates foralkaline phosphatase were added to the bag and distributed over theentire membrane for 2-3 minutes. The CSPD-treated membranes wereincubated at 37° C. for 30 minutes before an initial exposure to XRPX-ray film (Kodak) for 60 minutes. CDPSTAR reagent-treated membranes didnot require preincubation, and initial exposures were for 10 minutes.Exposure times were adjusted as necessary for resolution and clarity.The results are shown in FIG. 41.

In FIG. 41, the lane marked “M” contains molecular weight markers. Lanes1-5 contain reaction products from reactions run in the absence of KCl.Lane 1 contains the a reaction run without enzyme at 75° C. Lanes 2-5contain reaction products from reactions run at 60° C., 65° C., 70° C.and 75° C., respectively. Lanes 6-10 contain reaction products fromreactions run in the presence of 50 mM KCl. Lane 6 contains the areaction run without enzyme at 75° C. Lanes 7-10 contain reactionproducts from reactions run at 60° C., 65° C., 70° C. and 75° C.,respectively.

In general, a preferred pattern of cleavage products was produced whenthe reaction included 50 mM KCl. As seen in Lanes 7-10, the reactionproducts are more widely spaced in the 50 mM KCL-containing reactions atevery temperature tested as compared to the reactions run in the absenceof KCL (lanes 2-5; more of the cleavage products are found clustered atthe top of the gel near the uncut substrate). As seen in Lane 7 of FIG.41, cleavage reactions performed in 50 mM KCl at 60° C. produced apattern of cleavage products in which the products are maximally spreadout, particularly in the upper portion of the gel, when compared toother reaction condition patterns.

From the results obtained in this experiment, the optimal cleavageconditions for the 536 nucleotide sense strand fragment derived from thewild-type β-globin gene (SEQ ID NO:69) were determined to be 1×CFLPbuffer containing 1 mM MnCl₂ and 50 mM KCl at 60° C.

c) Optimization of the Cleavage Reaction Using Two Mutant Beta-GlobinSubstrates

From the results obtained above in a) and b), 60° C. was chosen as theoptimum temperature for the cleavage reaction when a β-globin substratewas to be used. When the wild-type substrate was utilized, running thecleavage reaction in the presence of 50 mM KCl generate the optimalpattern of cleavage products. The effect of varying the concentration ofKCl upon the cleavage pattern generated when both wild-type and mutantβ-globin substrates were utilized was next examined to determine theoptimal salt concentration to allow a comparison between the wild-typeand mutant β-globin substrates.

Single stranded substrates, 536 nucleotides in length, corresponding tomutant 1 (SEQ ID NO:71) and mutant 2 (SEQ ID NO:72) mutations wereprepared as described above in a). These two mutants each differ fromthe wild-type sequence by 1 nucleotide; they differ from each other by 2nucleotides.

For each substrate tested, 39 μl of a master mix containing DNA, CFLPbuffer and MnCl₂ was prepared. These mixes each included approximately500 fmoles of single-stranded, 5′ biotinylated 536 nucleotide substrateDNA, 39 μl of 1×CFLP buffer containing 1.54 mM MnCl₂ (giving a finalconcentration of 1 mM MnCl₂). The mixes were distributed into labeledreaction tubes in 6.5 μl aliquots. Each aliquot then received 0.5 μl of200 mM KCl for each 10 mM final KCl concentration (e.g., 2.0 μl added tothe 40 mM reaction tube) and all volumes were brought to 9 μl with dH₂O,No oil overlay was used. The reactions were brought to 95° C. for 5seconds, then cooled to 65° C. The reactions were then startedimmediately by the addition of 50 ng of the enzyme CLEAVASE BN in 1 μlof enzyme dilution buffer (20 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.5% NP40,0.5% TWEEN 20, 10 μg/ml BSA). All reactions proceeded for 5 minutes, andwere stopped by the addition of 8 μl of stop buffer. Samples were heatedto 72° C. for 2 minutes and 5 μl of each reaction was resolved byelectrophoresis through a 6% polyacrylamide gel (19:1 cross-link), with7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described above. The DNA was transferred to themembrane and the membrane was treated as described above in b) and thenexposed to X-ray film. The resulting autoradiograph is shown in FIG. 42.

In FIG. 42, the lane marked “M” contains molecular weight markers. Lanes1, 3, 5, 7, 9 and 11 contain reaction products from cleavage reactionsusing the mutant 1 substrate in the presence of 0, 10, 20, 30, 40 or 50mM KCl, respectively. Lanes 2, 4, 6, 8, 10 and 12 contain reactionproducts from cleavage reactions using the mutant 2 substrate in thepresence of 0, 10, 20, 30, 40 or 50 mM KCl, respectively.

FIG. 42 shows that while the pattern of cleavage products generated fromeach mutant changes as the KCl concentration is increased, distinctpatterns are generated from each mutant and differences in bandingpatterns are seen between the two mutants at every concentration of KCltested. In the mid-salt ranges (10 to 20 mM KCl), the larger cleavagebands disappear and smaller molecular weight bands appear (lanes 3-6).At higher salt concentrations (30 to 50 mM KCl), the larger molecularweight cleavage bands reappear with the cominant loss of the lowmolecular weight bands (lanes 7-12). Reaction conditions comprising theuse of 50 mM KCl were chosen as optimal from the results show in FIG.42. Clear differences in the intensities of a band appearing about 200nucleotides (see arrow in FIG. 42) is seen between the two mutantsubstrates under these reaction conditions.

d) The Enzyme CLEAVASE Generates Unique Cleavage Products from Wild-Typeand Mutant Beta-Globin Substrates

From the experiments performed above, the optimal reaction conditionswhen the wild-type or mutant 13-globin substrates were determined to bethe use of 50 mM KCl and a temperature of 60° C. These conditions werethen used to allow the comparison of the cleavage patterns generatedwhen the wild-type substrate (SEQ ID NO:69) was compared to the mutant 1(SEQ ID NO:71), mutant 2 (SEQ ID NO:72) and mutant 3 (SEQ ID NO:70)substrates.

Single-stranded substrate DNA, 534 or 536 nucleotides in length, wasprepared for the wild-type, mutant 1, mutant 2 and mutant 3 β-globingenes as described above in a). Cleavage reactions were performed asfollows. Reaction tubes were assembled which contained approximately 100fmoles of each DNA substrate in 9 μl of 1.1×CFLP buffer (1× finalconcentration) with 1.1 mM MnCl₂ (1 mM final concentration) and 55.6 mMKCl (50 mM final concentration). A “no enzyme” or uncut control was setup for each substrate DNA. The uncut controls contained one third asmuch DNA (approximately 33 fmoles) as did the enzyme-containingreactions to balance the signal seen on the autoradiograph.

The tubes were heated to 95° C. for 5 sec, cooled to 60° C. and thereactions were started immediately by the addition of 1 μl of the enzymeCLEAVASE BN (50 ng per μl in 1× dilution buffer). The uncut controlsreceived 1 μl of 1× dilution buffer.

Reactions proceeded for 5 min and then were stopped by the addition of 8μl of stop buffer. The samples were heated to 72° C. for 2 min and 5 μlof each reaction was resolved by electrophoresis through a 6%polyacrylamide gel (19:1 cross-link), with 7 M urea, in a buffer of 45mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described above. The DNA was transferred to themembrane and the membrane was treated as described above in b) and thenexposed to X-ray film. The resulting autoradiograph is shown in FIG. 43.

In FIG. 43, two panels are shown. The first panel shows the reactionproducts from the above cleavage reactions; the uncut controls are shownin lanes 1-4 and the cleavage products are shown in lanes 5-6. Thesecond panel is a magnification of lanes 5-8 to better shown thedifferent banding patterns seen between the substrate DNAs in the upperportion of the gel.

In FIG. 43, the lanes marked “M” contain biotinylated molecular weightmarkers prepared as described in Example 10. Lanes 1-4 contain the uncutcontrols for the wild-type, mutant 1, mutant 2 and mutant 3 β-globinsubstrates, respectively. Lanes 5-8 contain the cleavage products fromthe wild-type, mutant 1, mutant 2 and mutant 3 substrates, respectively.

From the results shown in FIG. 43, the enzyme CLEAVASE BN generates aunique pattern of cleavage products from each β-globin substrate tested.Differences in banding patterns are seen between the wild-type and eachmutant; different banding patterns are seen for each mutant allowing notonly a discrimination of the mutant from the wild-type but also adiscrimination of each mutant from the others.

The results shown here for the β-globin gene and above for thetyrosinase gene demonstrate that the CLEAVASE enzyme reaction provides apowerful new tool for the detection of mutated genes.

Example 16 Treatment of RNA Substrates Generates Unique CleavagePatterns

The present invention contemplates 5′ nuclease cleavage of single- ordouble-stranded DNA substrates to generate a unique pattern of bandscharacteristic of a given substrate. The ability of the 5′ nucleaseactivity of the enzyme CLEAVASE BN to utilize RNA as the substratenucleic acid was next demonstrated. This experiment showed that RNA canbe utilized as a substrate for the generation of a cleavage patternusing appropriate conditions (Lowering of the pH to 6.5 from 8.2 toreduce manganese-mediated degradation of the RNA substrate). Theexperiment was performed as follows.

An RNA transcript internally labelled with biotin was produced to serveas the substrate. The plasmid pGEM3Zf (Promega) was digested with EcoRI.EcoRI cuts the plasmid once generating a linear template. An RNAtranscript 64 nucleotides in length (SEQ ID NO:73) was generated by SP6transcription of the linearized template using a RIBOPROBE GEMINI Systemkit from Promega, Corp.; the manufacturer's directions were followedwith the exception that 25% of the UTP in the reaction was replaced withbiotin-UTP (Boehringer Mannheim) to produce an internally labelledtranscript. Following the transcription reaction (1 hour at 37° C.), theDNA template was removed by treatment with RQ1 RNase-free DNAse (fromthe RIBOPROBE GEMINI kit and used according to the manufacturer'sinstructions) and the RNA was collected and purified by precipitatingthe sample twice in the presence of 2 M NH₄OAc and ethanol. Theresulting RNA pellet was rinsed with 70% ethanol, air dried andresuspended in 40 μl of 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA.

Cleavage reactions contained 1 μl of the above RNA substrate and 50 ngof the enzyme CLEAVASE BN in 10 μl of 1×RNA-CFLP buffer (10 mM MOPS, pH6.3) and 1 mM of either MgCl₂ or MnCl₂. The reactions were assembledwith all the components except the enzyme and were warmed to 45° C. for30 sec. Reactions were started by the addition of 50 ng of the enzymeCLEAVASE BN in 1 μl of dilution buffer (20 mM Tris-HCl, pH 8.0, 50 mMKCl, 0.5% NP40, 0.5% TWEEN 20, 10 μg/ml BSA). Reactions proceeded for 10min and were stopped by the addition of 8 μl of stop buffer. The sampleswere heated to 72° C. for 2 minutes and 5 μl of each reaction wereresolved by electrophoresis through a 10% polyacrylamide gel (19:1cross-link), with 7 M urea, in a buffer containing 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1.2× SEQUENASE IMAGESBlocking Buffer, treated with 1×SAAP buffer and reacted withLUMIPHOS-530 (United States Biochemical) or Quantum YieldChemiluminescent Substrate (Promega) and exposed to X-ray film asdescribed in Example 10b. The resulting autoradiograph is shown in FIG.44.

In FIG. 44, lanes marked “M” contain molecular weight markers. Lane 1contains the no enzyme control and shows the migration of the uncutsubstrate. Lanes 2 and 3 contain reaction products from the incubationof the RNA substrate in a buffer containing MgCl₂ in the presence orabsence of the enzyme CLEAVASE BN, respectively. Lanes 4 and 5 containreaction products from the incubation of the RNA substrate in a buffercontaining MnCl₂ in the presence or absence of the enzyme CLEAVASE BN,respectively. A pattern of cleavage products is seen when the enzyme isincubated with the RNA substrate in the presence of MnCl₂, (lane 5).

These results show that the enzyme CLEAVASE can be used to probe RNAsubstrates for changes in sequence (i.e., point mutations, deletions,substitutions). This capability enables the examination of genes whichhave very large introns (e.g., greater than 10 kb) interrupting thecoding sequences. The spliced RNA transcript represents a simpler targetfor the scanning for mutations. In addition, the structural (i.e.,folding) information gained by cleavage of the RNA would be useful intargeting of hybridization or ribozyme probes to unstructured regions ofRNAs. Furthermore, because the cleavage reaction occurs so quickly, theenzyme CLEAVASE can be used to study various types of RNA folding andthe kinetics with which this folding occurs.

Example 17 The 5′ Nuclease Activity from Both CLEAVASE BN Enzyme And TaqPolymerase Generates Unique Cleavage Patterns Using Double-Stranded DNASubstrates

The ability of both the enzyme CLEAVASE BN and Taq polymerase togenerate cleavage patterns on single-stranded DNA templates wasexamined. The substrates utilized in this experiment were the 378nucleotide fragment from either the wild-type (SEQ ID NO:56) or 422mutant (SEQ ID NO:57) tyrosinase gene. These single-stranded substrateswere generated as described in Example 12a.

Cleavage reactions were performed as described in Example 12b with theexception that half of the reactions were cut with the enzyme CLEAVASEBN as described and a parallel set of reaction was cut with Taqpolymerase. The Taq polymerase reactions contained 1.25 units of Taqpolymerase in 1×CFLP buffer. The reaction products were resolved byelectrophoresis and the autoradiograph was generated as described inExample 12b. The autoradiograph is shown in FIG. 45.

In FIG. 45, lanes marked “M” contain biotinylated molecular weightmarkers. Lanes 1 and 2 contain the wild-type or 422 mutant substratecleaved with the enzyme CLEAVASE BN, respectively. Lanes 3 and 4 containthe wild-type or 422 mutant substrate cleaved with Taq polymerase,respectively.

FIG. 45 shows that both the enzyme CLEAVASE BN and Taq polymerasegenerate a characteristic set of cleavage bands for each substrateallowing the differentiation of the wild-type and 422 mutant substrates.The two enzyme produce similar but distinct arrays of bands for eachtemplate.

These results show that the 5′ nuclease of both the enzyme CLEAVASE BNand Taq polymerase are useful for practicing the cleavage reaction ofthe invention. Cleavage with Taq polymerase would find application whensubstrates are generated using the PCR and no intervening purificationstep is employed other than the removal of excess nucleotides usingalkaline phosphatase

Example 18 Multiplex Cleavage Reactions

The above Examples show that the cleavage reaction can be used togenerate a characteristic set of cleavage products from single-strandedDNA and RNA substrates. The ability of the cleavage reaction to utilizedouble-stranded DNA templates was examined. For many applications, itwould be ideal to run the cleavage reaction directly upon adouble-stranded PCR product without the need to isolate asingle-stranded substrate from the initial PCR. Additionally it would beadvantageous to be able to analyze multiple substrates in the samereaction tube (“multiplex” reactions).

Cleavage reactions were performed using a double-stranded template whichwas carried a 5′ biotin label on the sense-strand and a 5′ fluoresceinlabel on the anti-sense strand. The double-stranded substrate wasdenatured prior to cleavage. The double-stranded substrate was cleavedusing Taq polymerase. Taq polymerase was used in this experiment becauseit has a weaker duplex-dependent 5′ to 3′ exonuclease activity than doesthe enzyme CLEAVASE BN and thus Taq polymerase does not remove the 5′end label from the re-natured DNA duplexes as efficiently as does theenzyme CLEAVASE BN; therefore less signal is lost in the reaction.

The substrate utilized was a 157 by fragment derived from either thewild-type (SEQ ID NO:47), 419 mutant (SEQ ID NO:54) or 422 mutant (SEQID NO:55) of the tyrosinase gene. The wild-type fragment was generatedas described in Example 10a, the 419 mutant fragment was generated asdescribed in Example 10a and the 422 mutant fragment was generated asdescribed in Example 11 using PCR. The sense strand primer (SEQ IDNO:42) contains a 5′ biotin label and the anti-sense primer (SEQ IDNO:43) contains a 5′ fluorescein label resulting in the generation of adouble-stranded PCR product label on each strand with a different label.The PCR products were gel purified as described in Example 10a.

The cleavage reactions were performed as follows. Reaction tubes wereassembled with approximately 100 fmoles of the double-stranded DNAsubstrates in 5 μl of 1×CFLP buffer, 1 mM MnCl₂. The solutions wereoverlaid with a drop of mineral oil. The tubes were heated to 95° C. for30 sec and 1 unit of Taq polymerase (Promega) was added. Uncut controlscontained 33 fmoles of double-stranded DNA substrates in 5 μl of 1×CFLPbuffer, 1 mM MnCl₂. The reactions were cooled to 65° C. and incubated atthis temperature for 15 minutes. The reactions were stopped by theaddition of 8 μl of stop buffer. The samples were heated to 72° C. for 2min and 5 μl of reaction were resolved by electrophoresis through a 10%polyacrylamide gel (19:1 cross-link), with 7 M urea in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The entire set ofreactions was loaded in duplicate on the gel such that duplicate nylonmembranes containing the full set of reactions were created. Aftertransfer to a nylon membrane (performed as described in Example 10a),the membrane was cut in half; one half was probed using astreptavidin-alkaline phosphatase conjugate to visualize thebiotinylated sense-strand products (as described in Example 10a). Theother half of the membrane was probed with an anti-fluoresceinantibody-alkaline phosphatase conjugate to visualize thefluorescein-labelled anti-sense strand products (as described in Example5). The blots were visualized using the chemiluminescent proceduresdescribed in Examples 10a and 5 for biotin-labeled orfluorescein-labeled DNA, respectively. The autoradiographs are shownside-by side in FIG. 46.

In FIG. 46, the lane labeled “M1” contains biotinylated molecular weightmarkers prepared as described in Example 10a. The lane labeled “M2”contains molecular weight markers generated by digestion of pUC19 withMspI, followed by Klenow treatment to fill-in the ends. The blunt endswere then labeled with fluoresceinated dideoxynucleotides (BoehringerMannheim) using terminal transferase (Promega). Lanes M1 and 1-6 weredeveloped using the protocol for biotinylated DNA. Lanes 7-12 and M2were developed using the protocol for fluorescein-labeled DNA. Note thatin all lanes both strands of the substrate are present; only one strandis visualized in a given development protocol.

In FIG. 46, lanes 1-3 and 7-9 contain the “no enzyme” or uncut controlsusing the wild-type, 419 or 422 mutant substrates, respectively. Lanes4-6 and 10-12 contain cleavage products from the wild-type, 419 or 422mutant substrates, respectively. Unique patterns of cleavage productsare seen for each strand of each of the three substrates examined. Thus,a single reaction allowed the generation of a unique fingerprint fromeither the sense or anti-sense strand of each of the three tyrosinasefragments tested.

The results shown in FIG. 46 demonstrate that a cleavage pattern can begenerated from a double-stranded DNA fragment by denaturing the fragmentbefore performing the cleavage reaction. Note that in FIG. 46 thecleavage patterns were generated in the course of a single round ofheating to denature and cooling to cleave and that much of the substrateremains in an uncut form. This reaction would be amenable to performingmultiple cycles of denaturation and cleavage in a thermocycler. Suchcycling conditions would increase the signal intensity seen for thecleavage products. Substrates generated by the PCR performed in thestandard PCR buffer (50 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂,0.01% gelatin) can be treated to remove remaining dNTPs (e.g., additionof alkaline phosphatase) and to provide Mn²⁺. Under these conditions theCLEAVASE enzyme reaction can be performed on both strands of one or moreproducts generated in that PCR. Such a protocol reduces samplepreparation to a minimum resulting in a savings of both time andexpense.

The above example also demonstrates that two distinct substrates can beanalyzed in a single reaction thereby allowing the “multiplexing” of thecleavage reaction.

Example 19 Optimization of Manganese Ion Concentration for Cleavage ofDouble Stranded DNA Substrates

As discussed above, it may be desirable to run the cleavage reaction ondouble-stranded DNA substrates such restriction fragments or segmentsgenerated by balanced or symmetric PCR. The effect of varying theconcentration of Mn²⁺ in cleavage reactions using double-stranded DNAsubstrates was investigated. The results shown below demonstrate thatthe optimal concentration of Mn²⁺ is lower when a double-stranded DNAsubstrate is employed in the cleavage reaction as compared tosingle-stranded DNA substrates.

Two double-stranded (ds) DNA substrates, 157 by in length, derived fromthe tyrosinase mutants 419 (SEQ ID NO:40) and 422 (SEQ ID NO:84) wereprepared by PCR amplification of the exon 4 region of human tyrosinasegene as described above in Example 18. The sense strand of the 419 and422 tyrosinase mutant substrates contained a biotin-labeled at the 5′end following the PCR. The PCR products were gel purified as describedin Example 10a.

The cleavage reactions were performed as follows. Reaction tubes wereassembled with approximately 40 fmoles of the ds DNA substrates in 10 μlof water. The tubes were brought to 95° C. for 10 seconds in a PTC-100Programmable Thermal Controller (MJ Research, Inc.) to denature the DNA.The cleavage reactions were started by the addition of 10 μl of 2×CFLPbuffer (pH 8.2) containing 1 μl of the enzyme CLEAVASE BN (25 ng in 1×dilution buffer) and different concentrations of MnCl₂ such that thefinal concentration of MnCl₂ in reaction mixture (20 μl final volume)was either 0.5 mM, 0.25 mM, 0.15 mM, 0.1 mM, 0.05 mM and 0 mM. Aftermixing, the samples were immediately cooled to 65° C. and incubated atthis temperature for 5 minutes. The reactions were terminated by placingthe samples on ice and adding 10 μl of stop buffer. The samples wereheated to 85° C. for 30 sec and 10 μl of each reaction were resolved byelectrophoresis through a 10% polyacrylamide gel (19:1 cross-link), with7 M urea in a buffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10a. The DNA was transferred tothe membrane and the membrane was dried, washed in 1× SEQUENASE IMAGESBlocking Buffer (USB), treated with 1×SAAP buffer and reacted withCDPSTAR reagent (Tropix) and exposed to X-ray film as described inExample 10a. The resulting autoradiograph is shown in FIG. 47.

In FIG. 47, the lane marked “M” contains molecular weight markersprepared as described in Example 10. Lanes 1-6 contain the cleavageproducts generated by cleavage of the 419 mutant and lanes 7-12 containthe cleavage products generated by cleavage of the 422 mutant. Thereaction products generated by cleavage of the ds DNA substrates in1×CFLP buffer containing 0.5 mM (lanes 1,7); 0.25 mM (lanes 2,8); 0.15mM (lanes 3,9); 0.1 mM (lanes 4,10); 0.05 mM (lanes 5,11) and 0 mM MnCl₂(lanes 6,12) are shown.

The results shown in FIG. 47 show no cleavage is seen in the absence ofdivalent cations as is also the case for cleavage of ss DNA substrates(see Example 13(a) and FIG. 34). Optimal cleavage (i.e., production ofthe most distinct pattern of clevage fragments) of ds DNA substrates wasseen in the presence of 0.25 mM MnCl₂. This optimum is considerablylower than that obtained using ss DNA substrates (Example 13 and FIG. 34show that cleavage of ss DNA substrates was optimal in 1 mM MnCl₂.).FIG. 47 shows that the efficiency of cleavage of ds DNA substratesdecreases as the concentration of MnCl₂ is lowered; this effect islikely due to the lower efficiency of the enzyme in decreasingconcentrations of MnCl₂.

FIG. 47 shows that the cleavage pattern for dsDNA substrates apparentlydisappears when high concentrations of MnCl₂ (0.5 mM, lanes 1 and 7) areemployed in the cleavage reaction. This result is in contrast to theresults obtained when cleavage reactions are performed onsingle-stranded DNA (ssDNA) substrates. Example 13(a) showed thatefficient cleavage of ss DNA substrates were obtained in 1 mM MnCl₂ andlittle change in the cleavage pattern was seen when the Mn²⁺concentration varied from 1 to 4 mM.

The loss of the signal seen when ds DNA substrates are cleaved inbuffers containing high concentrations of MnCl₂ may be explained asfollows. The presence of high concentrations of divalent ions promotesthe reannealling of the DNA strands of the ds substrate during thecourse of the cleavage reaction. The enzyme CLEAVASE BN can nibble dsDNA substrates from the 5′ end (i.e., the enzyme removes short DNAfragments from the 5′ end in an exonucleolytic manner; see Example 6).This nibbling results in the apparent removal of the label from thesubstrate DNA (as the DNA contains a 5′ end label). Very short DNAfragments which contain the 5′ end label are not visualized as they runout of the gel or are not efficiently transferred to the membrane.

Example 20 Detection of Cleavage Patterns can be Automated

The ability to detect the characteristic genetic fingerprint of anucleic acid substrate generated by the cleavage reaction usingfluorescently labelled substrates in conjunction with automated DNAsequencing instrumentation would facilitate the use of the CFLP methodin both clinical and research applications. This example demonstratesthat differently labelled isolates (two different dyes) can be cleavedin a single reaction tube and can be detected and analyzed usingautomated DNA sequencing instrumentation.

Double-stranded DNA substrates, which contained either the N-hydroxysuccinimidyl ester JOE-NHS (JOE) or FAM-NHS (FAM) on the sense-strand,were generated using the PCR and primers labelled with fluorescent dyes.The anti-sense strand contained a biotin label. The substrates utilizedin this experiment were the 157 by fragments from the wild-type (SEQ IDNO:40) and 422 mutant (SEQ ID NO:55) of exon 4 of the tyrosinase gene.

The wild-type and 422 mutant tyrosinase gene substrates were amplifiedfrom cDNA plasmid clones containing either the wild-type (pcTYR-N1Tyr,Bouchard, B., et al. (1989) J. Exp. Med. 169:2029) or the 422 mutant(pcTYR-A422, Giebel, L. B., et al. (1991) 87:1119) forms of thetyrosinase gene. Each double-stranded substrate was amplified and the 5′ends labelled with either a biotin moiety or a fluorescent dye by usingthe following primer pairs in the PCR. The anti-sense primer of SEQ IDNO:43 containing a 5′-biotin moiety was obtained from Integrated DNATechnologies, Inc. (IDT, Coralville, Iowa). The biotinylated anti-senseprimer was used to prime the synthesis of both the wild-type and 422mutant substrates. The sense primer of SEQ ID NO:42 labelled with JOEwas used to prime synthesis of the wild-type tyrosinase gene. The senseprimer of SEQ ID NO:42 labelled with FAM was used to prime synthesis ofthe 422 mutant tyrosinase gene. The JOE and FAM-labelled primers wereobtained from Genset (Paris, France).

The PCR reactions were carried out as follows. One to two nanograms ofplasmid DNA from the wild-type or 422 mutant were used as the target DNAin a 100 μl reaction containing 50 μM of each dNTP, 1 μM of each primerin a given primer pair, 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl,0.05% TWEEN 20 and 0.05% NONIDET P-40. Tubes containing the abovemixture were overlaid with 70 μl Chill Out 14™ liquid wax (MJ Research,Watertown, Mass.). The tubes were heated to 95° C. for 1 min and thencooled to 70° C. Taq DNA polymerase (Perkin-Elmer) was then added as 2.5units of enzyme in 5 μl of a solution containing 20 mM Tris-Cl, pH 8.3,1.5 mM MgCl₂, 50 mM KCl, 0.05% TWEEN 20 and 0.05% NONIDET P-40. Thetubes were heated to 95° C. for 45 sec, cooled to 50° C. for 45 sec,heated to 72° C. for 1 min and 15 sec for 35 repetitions. Following thelast repetition, the tubes were incubated at 72° C. for 5 min.

The PCR products were gel purified as follows. The products wereresolved by electrophoresis through a 6% polyacrylamide gel (29:1cross-link) in a buffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mMEDTA. The DNA was visualized by ethidium bromide staining and the 157 byfragments were excised from the gel. The DNA was eluted from the gelslices by passive diffusion overnight into a solution containing 0.5 MNH₄OAc, 0.1% SDS and 0.1 M EDTA. The DNA was then precipitated withethanol in the presence of 4 μg of glycogen carrier. The DNA waspelleted and resuspended in 30 μl of H₂O.

The cleavage reactions were performed as follows. Approximately 100fmoles of each double-stranded DNA substrate (1-3 μl of each gelpurified DNA) in a total volume of 6 μl in H₂O was placed in a 500 μlthin wall microcentrifuge tube (Perkin-Elmer). The tube was heated to95° C. for 10 seconds to denature the substrates and then the tube wasquickly cooled to 50° C. (this step allows the DNA to assume its uniquesecondary structure by allowing the formation of intra-strand hydrogenbonds between complimentary bases). The cleavage reaction was started byadding 2 μl of 50 mM MOPS (pH 7.2), 1 μl of 1 mM MnCl₂ and 1 μl ofCLEAVASE BN enzyme (50 ng/μl). The cleavage reaction was performed in athermocycler (Perkin-Elmer DNA Thermal Cycler 480, Norwalk, Conn.)programmed to heat to 95° C. for 10 seconds and then cooled immediatelyto 50° C. The reaction was then incubated at 50° C. for 5 minutes andstopped by the addition of 1 μl of 10 mM EDTA.

Following the cleavage reaction, the sample was resolved by gelelectrophoresis using an ABI 373A DNA Sequencer (Foster City, Calif.).Prior to loading, the sample was denatured by adding 5 μl of a solutioncontaining 95% formamide and 10 mM EDTA and heating to 90° C. for 2minutes. Five microliters of the sample was resolved by electrophoresisthrough a 6% polyacrylamide gel (19:1 cross-link), with 6 M urea, in1×TBE buffer (89 mM Tris-Borate, pH 8.3, 2 mM EDTA). The gel was run at30 watts for 14 hours. Signals from four wavelength channels werecollected using the Applied Biosystem Data Collection program on aMACINTOSH computer. The raw data was analyzed with the BaseFinderprogram (Giddings, M., et al. (1993) Nucl. Acids Res. 21:4530) whichcorrects for the fluorescent spectrum overlap in the four channelsignals and mobility shifts caused by the use of different dye labels.

The results are shown in FIG. 48. FIG. 48 shows two traces representingthe two channel signals for the wild-type and mutant samples. Thewild-type sample, which was labeled with JOE dye, is shown by the thinlines. The mutant sample (R422Q), which was labeled with FAM dye, isshown by the thick lines. For comparison, a photograph of a highresolution polyacrylamide gel (10% gel with 19:1 crosslink) containingthe resolved cleavage products is shown above the traces (the top lanecontains cleavage fragments produced by clevage of the wild-typesubstrate; the bottom lane contains cleavage fragments produced byclevage of the R422Q mutant substrate). The cleavage products shown inthe gel, which contain biotin labels at the 5′ end of the sense strand,were generated, transferred to a nylon membrane and visualized asdescribed in Example 10a. Arrows point from selected bands seen uponcleavage of the 422 mutant substrate to the corresponding peaks in thetrace generated by the automated DNA sequencer (the arrows are labelled1 through 7 beginning with the left-hand side of FIG. 48).

Comparison of the two traces shows that differences in the cleavagepatterns generated from the cleavage of the wild-type and 422 mutantsubstrates in the same reaction are detected using automated DNAsequencing instrumentation. For example, cleavage of the 422 substrategenerates a cleavage product of approximately 56 nucleotides which isnot seen when the wild-type substrate is cleaved. This 56 nucleotideproduct is seen as the peak depicted by arrow 6 in FIG. 48. FIG. 48shows that not only are new cleavage products generated by cleavage ofthe mutant substrate, but that the cleavage of certain structures isenhanced in the mutant substrate as compared to the wild-type substrate(compare the intensity of the peaks corresponding to arrows 2-5 in thewild-type and mutant traces). In addition, certain cleavage products areshared between the two substrates and serve as reference markers (seearrows 1 and 7).

The above results show that automated DNA sequencing instrumentation canbe used to detect the characteristic genetic fingerprint of a nucleicacid substrate generated by the cleavage reaction. The results alsodemonstrate that the cleavage reaction can be run as a multiplexreaction. In this experiment both the wild-type and the mutant ds DNAsubstrates were cleaved in the same reaction (i.e., a multiplexreaction) and then were resolved on the same electrophoretic run usingan automated DNA sequencer.

Example 21 Identification of Viral Strains Using the CLEAVASE EnzymeReaction

The above examples demonstrate that the CLEAVASE enzyme reaction couldbe used to detect single base changes in fragments of varying size fromthe human β-globin and tyrosinase genes. These examples showed theutility of the CLEAVASE enzyme reaction for the detection andcharacterization of mutations in the human population. The ability ofthe CLEAVASE enzyme reaction to detect sequence variationscharacteristic of different strains of a virus was next examined.

The simian immunodeficiency virus (SIV) infection of monkeys is a widelyused animal model for the transmission of human immunodeficiency virustype-1 (HIV) in humans. Biological isolates of SIV contain multiplevirus strains. When a monkey is infected with a biological isolate ofSIV, unique subsets of the virus stock are recovered from the infectedanimals (specific strains are also able to infect tissue culture cells).Different genotypes of the virus are isolated from infected animalsdepending on the route of infection (Trivedi, P. et al. Journal ofVirology 68:7649 (1994)). The SIV long terminal repeat (LTR) containssequences which vary between the different viral strains and can be usedas a marker for the identification of the viral genotype.

In order to develop a rapid method for the identification of viralstrain(s) in a sample (e.g., a clinical isolate), the CLEAVASE enzymereaction was used to characterized SIV genotypes isolated afterinfection of cultured cells in vitro or after infection of rhesusmonkeys by either intravenous or intrarectal inoculation with unclonedbiological SIV stocks. Six clones generated from viral DNA isolatedfollowing in vitro infection of the CEMx174 cell line (L.CEM/251/12clone), after intravenous inoculation of monkeys (L100.8-1 clone), afterintrarectal low-dose inoculation of monkeys (L46.16-10 and L46.16-12clones) and after intrarectal high-dose inoculation of monkeys (L19.16-3and L36.8-3 clones) were obtained from C. David Pauza (Wisconsin PrimateResearch Center, Madison, Wis.). These clones were generated asdescribed by Trivedi, P. et al. Journal of Virology 68:7649 (1994).These plasmid clones contained viral LTR sequences and were utilized togenerate double-stranded DNA (ds DNA) substrates for the cleavagereaction.

a) Preparation of the Substrate DNA

The six SIV plasmids were utilized as templates in PCRs in order togenerate dsDNA substrates for the cleavage reaction. The primer pairutilized spans the U3-R boundary in the SIV LTR and amplifies anapproximately 350 by fragment. This portion of the SIV LTR containsrecognition sequences for transcription factors (including Sp1 andNF-κB) as well as the TATA box for transcription initiation and ispolymorphic in different viral strains (Trivedi, P. et al., supra).

The primer pair consisting of SEQ ID NOS:74 and 75 was used to amplifythe SIV LTR clones in the PCR. SEQ ID NO:74 primes synthesis of the (+)strand of the SIV LTR and comprises 5′-GGCTGACAAGAAGGAAACTC-3′. SEQ IDNO:75 primes synthesis of the (−) strand of the SIV LTR and comprises5′-CCAGGCGGCG GCTAGGAGAGATGGG-3′. To visualize the cleavage patterngenerated by cleavage of the (+) strand of the LTR, the PCR wasperformed using the primer consisting of SEQ ID NO: 74 containing abiotin label at 5′ end and unlabeled primer consisting of SEQ ID NO:75.To visualize the cleavage pattern generated by clevage of the (−) strandof the viral LTR, the PCR was performed using the primer pair consistingof SEQ ID NO:75 containing a biotin label at the 5′ end and unlabeledprimer SEQ ID NO:74.

The PCR reactions were carried out as follows. Ten to twenty nanogramsof plasmid DNA from each of the above 6 SIV LTR clones was used as thetarget DNA in separate 100 μl reactions containing 60 μM of each dNTP,0.2 μM of each primer in a given primer pair, 10 mM Tris-Cl, pH 9.0 (at25° C.), 2 mM MgCl₂, 50 mM KCl, with 0.1% TRITON X-100. Tubes containingthe above mixture were overlaid with two drops of light mineral oil andthe tubes were heated to 96° C. for 3 min and Taq DNA polymerase(Perkin-Elmer) was then added as 2.5 units of enzyme in 0.5 μl of 20 mMTris-HCl, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol and0.5% TWEEN 20 and 0.5% NONIDET P-40. The tubes were heated to 96° C. for45 seconds, cooled to 60° C. for 45 seconds, heated to 72° C. for 1minute for 35 repetitions. Following the PCR, the reaction mixture wasseparated from the mineral oil and 5 μl of 5M NaCl, 4 μl of 10 mg/mlglycogen and 250 μl of 100% ethanol were added to the aqueous PCRsamples. After incubation at −20° C. for 1 hour, the DNA was pelleted bycentrifugation in a MARATHON MICRO A centrifuge (Fisher Scientific) atmaximum speed for 5 minutes and resuspended in 40 μl of 10 mM Tris-HCl,pH 8.0, 0.1 mM EDTA.

The PCR products were gel purified as follows. The DNA was mixed with0.5 volume of loading buffer (95% formamide, 5 mM EDTA, 0.02% bromphenolblue, 0.02% xylene cyanol) and heated to 75° C. for 2 minutes. Theproducts were resolved by electrophoresis through a 6% polyacrylamidedenaturing gel (19:1 cross-link) in a buffer containing 7M urea, 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. The DNA was visualized by ethidiumbromide staining and the product bands were excised from the gel. TheDNA was eluted from the gel slices by passive diffusion overnight into asolution containing 0.5 M NH₄OAc, 0.1% SDS and 0.1 M EDTA. The DNA wasthen precipitated with ethanol in the presence of 4 μg of tRNA carrier.The DNA was pelleted and resuspended in 50 μl of 0.2 M NaCl, 10 mMTris-HCl, pH8.0, 0.1 mM EDTA. The DNA was precipitated with ethanol andresuspended in 50 μl of 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA. The finalDNA concentration was estimated to be 40 fmole/μl for eachdouble-stranded SIV LTR PCR product.

b) DNA Sequence Analysis Of The SIV LTR PCR Products

The DNA sequence of the six PCR fragments generated in section a) abovewas determined using the FMOL DNA Sequencing System (Promega) accordingto the manufacturer's instructions. For each set of the sequencingreactions 0.2 pmoles of the PCR product and 2 pmoles of one of the two5′-biotinylated PCR primers SEQ ID NOS:74 and 75 was used (i.e., bothstrands of the PCR fragments were sequenced). Following the sequencingreactions, the sequencing products were resolved by electrophoresis.After electrophoresis, the DNA bands were visualized following transferto a nylon membrane as described in Example 19 with the followingmodification. A solution containing 0.2% Blocking reagent(Boehringer-Mannheim) and 0.2% SDS in TBS buffer (100 mM Tris-HCl,pH7.4; 68 mM NaCl) was used in place of the 1× SEQUENASE IMAGES BlockingBuffer (USB).

The sequence of the 351 by fragment derived from the L100.8-1 LTR cloneis listed in SEQ ID NO:76. The sequence of the 340 by fragment from theL46.16-10 LTR clone is listed in SEQ ID NO:77. The sequence of the 340by fragment derived from the L46.16-12 LTR clone is listed in SEQ IDNO:78. The sequence of the 351 by fragment from the L19.16-3 LTR cloneis listed in SEQ ID NO:79. The sequence of the 351 by fragment derivedfrom the LCEM/251/12 LTR clone is listed in SEQ ID NO:80. The sequenceof the 351 by fragment derived from the L36.8-3 LTR clone is listed inSEQ ID NO:81.

Analysis of sequenced LTR fragments shows that they have multiplesubstitutions and a deletion relative to the L100.8-1 LTR sequence (SEQID NO:76); the L100.8-1 LTR sequence was chosen as a reference to permitcomparisons between the six LTR clones. For the ease of discussion, thefirst or 5′-terminal nucleotide of the (+) strand of L100.8-1 LTRsequence (SEQ ID NO:76) is defined as number 1 and the last or3′-terminal nucleotide of this sequence is defined as number 351.

FIG. 49 displays the nucleotide sequence of the six LTR clones. Thereference clone, L.100.8-1 (SEQ ID NO:76), is shown on the top line.Sequences appearing in bold type represent sequence changes relative tothe sequence of clone L.100.8-1 (SEQ ID NO:76). The sequences outlinedby the brackets in FIG. 49 represent palindromic sequences which canform a very stable hairpin structure having a stem of 14 base pairs(12/14 bases in the stem are complementary) and a loop of 7 nucleotidesin the reference clone L.100.8-1 (SEQ ID NO:76). This hairpin structureis present in all six LTR clones although the sequence of the stem andloop structures varies between the clones.

In comparison with L100.8-1 sequence (SEQ ID NO:76), the L46.16-10sequence (SEQ ID NO:77) has seven substitutions and one 11 nucleotidedeletion corresponding to nucleotides 65-75 of SEQ ID NO:76. Thesubstitutions are: C to T in position 28 (C28T), C57T, G90A, C97T,G238A, G242A and G313A. The L46.16-12 sequence (SEQ ID NO:78) has sevensubstitutions and one 11 nucleotide deletion corresponding tonucleotides 65-75 of SEQ ID NO:76. The substitutions are: C28T, C57T,G90A, C97T, A103G, G242A and G313A. L19.16-3 sequence (SEQ ID NO:79) hastwo substitutions: A94C and A317T. LCEM/251/12 sequence (SEQ ID NO:80)has seven substitutions: G26A, G72A, C97T, G258A, A281C, G313A andC316T. L36.8-3 sequence (SEQ ID NO:81) has six substitutions: G60A,G172A, G207A, G221A, T256C and C316T.

c) Cleavage Reaction Conditions and CFLP Analysis of The (−) Strand ofthe SIV LTR

Double-stranded substrates corresponding to the SIV LTR sequences listedin SEQ ID NOS:76-81 were labelled on the (−) strand using the PCR andthe primer pair corresponding to SEQ ID NO: 74 and 75. The primer of SEQID NO:75 (the (−) strand primer) contained a biotin label at the 5′ end.The PCR was performed and the reaction products were isolated asdescribed in section a).

The cleavage reactions were performed as follows. Reaction tubes wereassembled with approximately 60 fmoles of the ds DNA substrates in 6 μlof water. The following reagents were added to the DNA: 2 μl of 5×CFLPbuffer (pH 7.2) containing 150 mM KCl (to yield a final concentration of30 mM KCl) and 1 μl of the CLEAVASE BN enzyme (25 ng in 1× dilutionbuffer). A reaction tube containing the above components with theexception that 1 μl of H₂O was added in place of the CLEAVASE BN enzymewas prepared and run as the uncut or no enzyme control. The tubes werebrought to 95° C. for 10 seconds in a PTC-100 Programmable ThermalController (MJ Research, Inc.) to denature the DNA. Following thedenaturation step, the tubes were immediately cooled to 40° C. Thecleavage reaction was immediately started by the addition of 1 μl of 2mM MnCl₂ (to achieve a final concentration of 0.2 mM). The tubes wereincubated at 40° C. for 5 minutes. The reactions were terminated byadding 6 μl of stop buffer. The samples were heated to 85° C. for 30 secand 5 μl of each reaction were resolved by electrophoresis through a 12%polyacrylamide gel (19:1 cross-link), with 7 M urea in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10a. The DNA was transferred tothe membrane and the membrane was dried, washed in 0.2% Blocking reagent(Boehringer Mannheim); 0.2% SDS in 100 mM Tris-HCl, pH 7.4; 68 mM NaCl,treated with 1×SAAP buffer and reacted with CDPSTAR reagent (Tropix) andexposed to X-ray film as described in Example 10a. The resultingautoradiograph is shown in FIG. 50.

FIG. 50 shows the cleavage patterns which correspond to the cleavage ofthe (−) strand of the double-stranded LTR substrates. In FIG. 50, thelane marked “M” contains molecular weight markers (prepared as describedin Example 10). Lanes 1-6 contain the cleavage products generated bycleavage of the L100.8-1, L46.16-10, L46.16-12, L19.16-3, LCEM/251/12and L36.8-3 LTR PCR fragments, respectively. Lanes 7-12 contain theuncut controls of each of the 6 LTR substrates in the same order as thatdescribed for Lanes 1-6.

The results shown in FIG. 50 show that the cleavage or CFLP pattern foreach LTR substrate contains multiple bands which range in size fromapproximately 350 nucleotides (the uncut substrate) to less than 24nucleotides. The bands located below about 100 nucleotides in lengthshow differences between the six clones which reflect nucleotide changescharacteristic of the different SIV LTR isolates. Examination of theCFLP patterns revealed that the reaction detected five unique cleavagepatterns among the six SIV LTR isolates. From the DNA sequence data, itwas known that all six LTR clones were unique. However, the CFLP patternappeared to be identical for the clones shown in lanes 2 and 3.

The CFLP patterns generated by cleavage of the (−) strand from all sixsubstrates contain a strong band which corresponds to a fragment ofapproximately 100 nucleotides in length. This band corresponds tocleavage of all six LTR substrates at the long palindromic sequencelocated 97 nucleotides from the 5′ end of the (−) strand (see thebracketed region in FIG. 49). This palindromic sequence forms a verystable hairpin structure in single-stranded DNA and provides an optimalsubstrate for the CLEAVASE BN enzyme. Cleavage of this hairpin structureis predicted to generate a fragment of approximately 100 nucleotides.

The LTR substrates, L46.16-10 (SEQ ID NO:77) and L46.16-12 (SEQ IDNO:78), shown in lanes 2 and 3 were generated from the same animal usingthe same route of infection (Trivedi, P. et al., supra). Thesesubstrates have identical sequences in the region corresponding to thedetectable cleavage sites (i.e., below 100 nucleotides) with theexception of a single base; the L46.16-10 clone (SEQ ID NO:77) containsa G to A change at position 239 (G239A) relative to the referencesequence listed in SEQ ID NO:76. Examination of the DNA sequence ofthese two clones reveals that this substitution is located in the loopregion of a strong hairpin structure (see the palindromic regionbracketed in FIG. 49). Because the single base difference between thesetwo sequences is located in the loop region of the hairpin structure, itmay not change DNA secondary structure of the two substratessufficiently to generate different CFLP patterns under the conditionsutilized here. It may be possible to detect this single base differencebetween these two clones by varying the reaction conditions in a waythat destablizes the strong hairpin structure.

The results shown in FIG. 50 demonstrate that the CFLP reaction can beused to detect the majority (5/6 or 83%) of the sequence variationspresent in the six SIV LTR clones studied. In addition, FIG. 50demonstrates that the CFLP reaction is a sensitive means for probing thesecondary structure of single strands of nucleic acids.

d) Cleavage Reaction Conditions and CFLP Analysis of the (+) Strand ofthe SIV LTR

Double-stranded substrates corresponding to the SIV LTR sequences listedin SEQ ID NOS:76-81 were labelled on the (+) strand using the PCR andthe primer pair corresponding to SEQ ID NO: 74 and 75. The primer of SEQID NO:74 (the (+) strand primer) contained a biotin label at the 5′ end.The PCR was performed and the reaction products were isolated asdescribed in section a). The cleavage reactions, electrophoresis and DNAvisualization were performed as described above in section c). Theresulting autoradiograph is shown in FIG. 51.

FIG. 51 shows the resulting pattern corresponding to the cleavageproducts of the (+) strand of six SIV LTR fragments. The lane marked “M”contains molecular weight markers (prepared as described in Example 10).Lanes 1-6 contain the cleavage products generated by cleavage of theL100.8-1, L46.16-10, L46.16-12, L19.16-3, LCEM/251/12 and L36.8-3 LTRPCR fragments, respectively. Lanes 7-12 contain the uncut controls ofeach of the 6 LTR substrates in the same order as that described forLanes 1-6.

As was shown for cleavage of the (−) strand of the LTR clones, the CFLPpattern for each (+) strand of the SIV LTR substrates contains uniquefeatures that characterize the majority of specific nucleotidesubstitutions. For example, deletion of 11 nucleotides can be easilydetected for L46.16-10 (SEQ ID NO:77) and L46.16-12 (SEQ ID NO:78) (FIG.51, lanes 2 and 3). This deletion removes one of the three Sp1 bindingsites and is a change characteristic of the genotype of SIV whichpredominates in animals which are infected using low-doses of virusstock via intrarectal inoculation (Trivedi, P. et al., supra). The CFLPpattern generated by cleavage of the (+) strand of the substratesderived from clones L46.16-10 and L46.16-12 again were identical underthese reaction conditions.

The results shown above demonstrate that the CFLP reaction can be usedas a means to rapidly identify different strains (i.e., genotypes) ofvirus. The ability to rapidly identify the particular strain of virus orother pathogenic organism in a sample is of clinical importance. Theabove results show that the CFLP reaction can be used to provide a fastmethod of strain or species identification.

Example 22 The Effects of Alterations in Salt Conditions in CleavageReactions Using a Single-Stranded DNA Substrate

In Example 13 it was shown that the CLEAVASE enzyme reaction isinsensitive to large changes in reactions conditions when asingle-stranded DNA is employed as the substrate. Example 13 showed thatthe cleavage reaction can be performed using a range of saltconcentrations (0 to 50 mM KCl) in conjunction with single-strandedsubstrates. In this example, the effect of substituting other salts inplace of KCl was examined in cleavage reactions using single-strandedDNA substrates.

a) Effect of Substituting NaCl for KCl in Cleavage

Reactions Using A Single-Stranded Template

To determine the effect of substituting NaCl in place of KCl upon thecleavage pattern created by 5′ nuclease activity on a single-strandedDNA substrate, the following experiment was performed. A single templatewas incubated in the presence of a fixed amount of the CLEAVASE BNenzyme (50 ng) in a buffer containing 10 mM MOPS, pH 8.2, 1 mM MnCl₂ andvarious amounts of NaCl.

Approximately 100 fmoles of the 157 nucleotide fragment derived from thesense strand of exon 4 of the tyrosinase gene (SEQ ID NO 47; prepared asdescribed in example 10b) were placed in a 500 μl thin wallmicrocentrifuge tubes (Perkin Elmer, Norwalk, Conn.) in 1×CFLP buffer,pH 8.2 and 1.33 mM MnCl₂ (to yield a final concentration of 1 mM MnCl₂)in a volume of 15 μl. NaCl was added to yield a final concentration of0, 10, 20, 30, 40, 50, 75 or 100 mM. The final reaction volume was 20μl.

A tube containing 1×CFLP buffer, pH 8.2, 1 mM MnCl₂ and 100 fmolessubstrate DNA was prepared and served as the no salt, no enzyme control(sterile distilled water was substituted for CLEAVASE BN enzyme and allreaction components were added prior to denaturation at 95° C.).

The tubes were heated to 95° C. for 20 seconds and then rapidly cooledto 65° C. The cleavage reaction was started immediately by the additionof 5 μl of a diluted enzyme mixture comprising 1 μl of CLEAVASE BNenzyme (50 ng/μl in 1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mMTris-HCl, pH 8.0, 50 mM KCl, 10 μg/ml BSA)) in 1×CFLP buffer, pH 8.2without MnCl₂.

After 5 minutes at 65° C., reactions were stopped by the addition of 16μl of stop buffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue,0.05% xylene cyanol). Samples were heated to 72° C. for 2 minutes and 7μl of each reaction were resolved by electrophoresis through a 10%polyacrylamide gel (19:1 cross-link), with 7M urea, in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, as described inExample 10a.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in example 10a. The DNA was transferred tothe membrane and the membrane was dried, blocked in 1× I-BLOCK (Tropix,Bedford, Mass.), conjugated with streptavidin-alkaline phosphatase(United States Biochemical), washed, reacted with CDPSTAR reagent(Tropix, Bedford, Mass.) as described in Example 10a with the exceptionthat 0.01 ml CDPSTAR reagent was added per cm² of membrane. The membranewas exposed to X-ray film as described in Example 10a. The results areshown in FIG. 52.

In FIG. 52, the lane marked “M” contains molecular weight markers asdescribed in example 10a. Lane 1 contains the no salt, no enzyme controland shows the mobility of the uncleaved template DNA. Lanes 2 through 9contain reaction products incubated in the presence of CLEAVASE BNenzyme in a buffer containing 0, 10, 20, 30, 40, 50 75 or 100 mM NaCl,respectively.

The results shown in FIG. 52 demonstrate that the substitution of NaClin place of KCl has little or no effect upon the cleavage patterngenerated using the 157 nucleotide tyrosinase DNA substrate (SEQ IDNO:47). Essentially the same dependence of the cleavage pattern on saltconcentration was observed using this single-stranded DNA substrate wheneither KCl (See example 13b, FIG. 35) or NaCl (FIG. 52) was employed inthe cleavage reaction.

b) Effect of Substituting (NH₄)₂SO₄ for KCl in Cleavage Reactions Usinga Single-Stranded Template

In an approach similar to that described in above in section a), theeffect of substituting (NH₄)₂SO₄ in place of KCl upon the cleavagepattern created by 5′ nuclease activity on a single-stranded DNAsubstrate was tested. Cleavage reactions were set up exactly asdescribed in section a) with the exception that variable amounts of(NH₄)₂SO₄ were used in place of the NaCl.

Approximately 100 fmoles of the 157 nucleotide fragment derived from thesense strand of exon 4 of the tyrosinase gene (SEQ ID NO 47; prepared asdescribed in example 10a) were placed in 500 μl thin wallmicrocentrifuge tubes (Perkin Elmer, Norwalk, Conn.) in 1×CFLP buffer,pH 8.2 and 1.33 mM MnCl₂ (to yield a final concentration of 1 mM) in avolume of 15 μl. (NH₄)₂SO₄ was added to yield a final concentration of0, 10, 20, 30, 40, 50, 75 or 100 mM. The final reaction volume was 20μl.

A tube containing 1×CFLP buffer, pH 8.2, 1 mM MnCl₂ and 100 fmolessubstrate DNA was prepared and served as the no salt, no enzyme control(sterile distilled water was substituted for CLEAVASE BN enzyme and allreaction components were added prior to denaturation at 95° C.).

The tubes were heated to 95° C. for 20 seconds and then rapidly cooledto 65° C. The cleavage reaction was started immediately by the additionof 5 μl of a diluted enzyme mixture comprising 1 μl of CLEAVASE BNenzyme (50 ng/ml in 1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mMTris-HCl, pH 8.0, 50 mM KCl, 10 μg/ml BSA)) in 1×CFLP buffer, pH 8.2without MnCl₂.

After 5 minutes at 65° C., reactions were stopped by the addition of 16μl of stop buffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue,0.05% xylene cyanol). Samples were heated to 72° C. for 2 minutes and 7μl of each reaction were resolved by electrophoresis through a 10%polyacrylamide gel (19:1 cross-link), with 7M urea, in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, as described inExample 10a.

After electrophoresis, the DNA was transferred to a membrane and thedetected as described in section a) above. The resulting autoradiographis shown in FIG. 53.

In FIG. 53, the lane marked “M” contains molecular weight markers asdescribed in example 10a. Lane 1 contains the no enzyme control andshows the mobility of the uncleaved template DNA. Lanes 2 through 9contain reaction products incubated in the presence of CLEAVASE BNenzyme in a buffer containing 0, 10, 20, 30, 40, 50 75 or 100 mM(NH₄)₂SO₄, respectively.

The results shown in FIG. 53 demonstrate that the cleavage reaction isseverely inhibited by the presence of (NH₄)₂SO₄. The reaction iscompletely inhibited by as little as 20 mM (NH₄)₂SO₄; the extent of thecleavage reaction in 10 mM (NH₄)₂SO₄ is comparable to that obtained in50 mM KCl or NaCl and is significantly reduced relative that obtained at0 mM (NH₄)₂SO₄. The pattern of cleavage obtained at 10 mM (NH₄)₂SO₄,however, is identical to that observed when the 157 nucleotide template(SEQ ID NO:47) incubated in the absence of (NH₄)₂SO₄ or in KCl or NaCl.This indicates that the choice of salt included in the CLEAVASE enzymereaction has no effect on the nature of the sites recognized assubstrates by the CLEAVASE BN enzyme (i.e., the inhibitory effect seenis due the effect of (NH₄)₂SO₄ upon enzyme activity not upon theformation of the cleavage structures).

c) Effect of Increasing KCl Concentration on the Cleavage ofSingle-Stranded Substrates

The effect of increasing the concentration of KCl in cleavage reactionsusing a single-stranded DNA substrate was examined by performing thecleavage reaction in concentrations of KCl which varied from 0 to 100mM. The cleavage reactions were performed as described in section a)with the exception that KCl was added to yield final concentrations of0, 25, 50, 75 or 100 mM and 200 fmoles of the substrate were used in thereaction; additionally the substrate DNA was denatured by incubation at95° C. for 5 seconds.

Following the cleavage reaction, the samples were electrophoresed,transferred to a membrane and detected as described in section a) above.The resulting autoradiograph is shown in FIG. 54.

In FIG. 54, the lanes marked “M” contains molecular weight markers asdescribed in Example 10a. Lane 1 is the no enzyme control; Lanes 2-7contain reactions carried out in the presence of 0, 25, 50, 75, 100 or100 mM KCl (the 100 mM sample was repeated twice), respectively.

The results shown in FIG. 54 demonstrate that the extent of cleavage inthe cleavage reaction decreased as a function of increasing KClconcentration (although residual cleavage was detectable at 100 mM KCl).Furthermore, the pattern of fragments generated by cleavage ofsingle-stranded substrates with CLEAVASE BN enzyme is unaffected by theconcentration of KCl present in the reactions.

d) Effect of High KCl Concentrations on Cleavage Reactions Using aSingle-Stranded Substrate

The ability of the CLEAVASE enzyme reaction to be carried out atrelatively high concentrations of KCl was tested by performing thecleavage reaction in the presence of variable concentrations of KCl inexcess of 100 mM. The reactions were performed using the 157 nucleotidefragment from exon 4 of the tyrosinase gene (SEQ ID NO:47) as describedabove in section c), with the exception that KCl was added to yield afinal concentration of 0, 100, 150, 200, 250 or 300 mM.

Following the cleavage reaction, the samples were electrophoresed,transferred to a membrane and detected as described in section a) above.The results (data not shown) indicated that the cleavage reaction wasseverely inhibited by KCl concentrations in excess of 100 mM. Someresidual cleavage did, however, persist at these elevated saltconcentrations, up to and including 300 mM KCl.

e) Effect of KCl Concentration on the Stability of the Cleavage PatternDuring Extended Incubation Periods

The results presented above demonstrate that the CLEAVASE enzymereaction is inhibited by elevated concentrations (i.e., above 50 mM) ofeither KCl or NaCl. To determine whether this inhibition wouldeffectively result in the stabilization of the cleavage pattern afterextended reaction times (i.e., due to inhibition of enzyme activity),reactions were examined at varying extended time points at both 0 and 50mM KCl.

Approximately 100 fmoles of the 157 nucleotide fragment derived from thesense strand of exon 4 of the tyrosinase gene (SEQ ID NO 47; prepared asdescribed in example 10a) were placed in 200 μl thin wallmicrocentrifuge tubes (BioRad, Richmond, Calif.) in 1×CFLP buffer, pH8.2, 1.33 mM MnCl₂ (to yield a final concentration of 1 mM) and KCl toyield a final concentration of 0 or 50 mM KCl. The final reaction volumewas 20 μl.

Control reactions which lacked enzyme were set up in parallel for eachtime point examined; these no enzyme controls were prepared as describedabove with the exception that sterile distilled water was substitutedfor CLEAVASE BN enzyme and all reaction components were added prior todenaturation at 95° C.

The tubes were heated to 95° C. for 20 seconds and then rapidly cooledto 65° C. The cleavage reaction was started immediately by the additionof 5 μl of a diluted enzyme mixture comprising 1 μl of CLEAVASE BNenzyme (50 ng/ml in 1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mMTris-HCl, pH 8.0, 50 mM KCl, 10 μg/ml BSA)) in 1×CFLP buffer, pH 8.2without MnCl₂. Twenty microliters of Chill Out 14™ (MJ Research,Watertown, Mass.) were added to each tube after the addition of theenzyme. The reactions were allowed to proceed at 65° C. for 5 min, 30min, 1 hour, 2 hours, 4 hours and 17 hours.

At the desired time point, the reactions were stopped by the addition of16 μl of stop buffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue,0.05% xylene cyanol). Samples were heated to 72° C. for 2 minutes and 7μl of each reaction were resolved by electrophoresis through a 10%polyacrylamide gel (19:1 cross-link), with 7M urea, in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, as described inExample 10a.

After electrophoresis, the DNA was transferred to a membrane and thedetected as described in section a) above. The resulting autoradiographis shown in FIG. 55.

In FIG. 55, the lane marked “M” contains molecular weight markers asdescribed in example 10a. Lanes 1-10 contain products from reactionscarried out in the absence of KCl; lanes 11-20 contain products fromreactions carried out in the presence of 50 mM KCl. Lanes 1, 3, 5, 7,and 9 contain no enzyme controls incubated for 5 minutes, 30 minutes, 1hour, 2 hours, 4 hours and 17 hours, respectively. Lanes 2, 4, 6, 8, and10 contain the reaction products from reactions incubated at 65° C. for5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 17 hours,respectively. Lanes 11, 13, 15, 17, and 19 contain no enzyme controlsincubated in 50 mM KCl for 5 minutes, 30 minutes, 1 hour, 2 hours, 4hours and 17 hours, respectively. Lanes 12, 14, 16, 18 and 20 containreaction products from CFLP reactions incubated in 50 mM KCl at 65° C.for 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 17 hours,respectively.

The results indicated that cleavage was retarded in the presence of 50mM KCl which resulted in a significant stabilization of the cleavagepattern (i.e., the cleavage pattern remained the same over time becausethe rate of cleavage was dramatically slowed and thus the largercleavage fragments are not further cleaved to produce smallerfragments). Note that at the extended incubation times, the reactionscarried out in the absence of KCl were significantly overdigested; after1 hour at 65° C., essentially no large fragments remain, and there issubstantial accumulation of small cleavage products. In contrast, thereactions carried out at 50 mM KCl were essentially static between 30minutes and 4 hours; overdigestion was only apparent at the longest timepoint and was not as extensive as that observed in the absence of KCl.

Example 23

Comparison of the Patterns of Cleavage Generated byCleavage-of-Single-Stranded and Double-Stranded DNA Substrates

In CLEAVASE BN enzyme-mediated primer-independent cleavage ofdouble-stranded DNA substrates, the two strands of DNA are separated ina denaturation step prior to the addition of enzyme. Therefore, thepatterns generated by cleaving double-stranded templates should beidentical to those generated by cleaving single-stranded template. Thisassumption was verified by the experiment described below.

The single-stranded substrate comprising the 157 nucleotide fragmentderived from the sense strand of exon 4 of the tyrosinase gene (SEQ IDNO:47) was prepared as described in example 10b with the followingmodification. After gel purification and precipitation in the presenceof glycogen carrier, the PCR products were resuspended in TE (10 mMTris-Cl, pH 8.0, 1 mM EDTA) and then reprecipitated with 2 M NH₄OAc and2.5 volumes of ethanol. The DNA was then resuspended in 400 μl of 10 mMTris-HCl, pH 8.0, 0.1 mM EDTA.

Approximately 50 or 100 fmoles of the single-stranded 157 nucleotidefragment (SEQ ID NO: 47) were placed in a 200 μl centrifuge tube(BioRad, Richmond, Calif.) in 1×CFLP buffer, pH 8.2 and 1.33 mM MnCl₂(final concentration was 1 mM MnCl₂) in a volume of 15 μl. The finalreaction volume was 20 μl. A 20 μl no salt, no enzyme control was set upin parallel; this reaction contained sterile distilled water in place ofthe CLEAVASE BN enzyme and all reaction components were added prior todenaturation at 95° C.

The reaction tubes were heated to 95° C. for 5 seconds and then rapidlycooled to 65° C. The cleavage reactions were started immediately by theaddition of 5 μl of a diluted enzyme mixture comprising 1 μl of CLEAVASEBN enzyme (50 ng/μl in 1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg/ml BSA)) in 1×CFLP buffer, pH 8.2without MnCl₂. After 5 minutes at 65° C., reactions were stopped by theaddition of 16 μl of stop buffer (95% formamide, 10 mM EDTA, 0.05%bromophenol blue, 0.05% xylene cyanol).

A double stranded form of the 157 nucleotide substrate was cleaved withCLEAVASE BN enzyme in the same experiment. This double-strandedsubstrate (SEQ ID NO:40) was generated as described in Example 10b withthe following modifications. After gel purification and precipitation inthe presence of glycogen carrier, the PCR products were resuspended inTE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA) and then reprecipitated with 2 MNH₄OAc and 2.5 volumes of ethanol. The DNA was then resuspended in 400μl of 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA.

Approximately 33 or 66 fmoles of the double-stranded 157 by fragment(SEQ ID NO:40) were placed in a 200 μl thin walled microcentrifuge tube(BioRad, Richmond, Calif.). Sterile distilled water was added to avolume of 15 μl.

The reaction tubes were heated to 95° C. for 5 seconds and then rapidlycooled to 65° C. The cleavage reactions were started immediately by theaddition of 5 μl of a diluted enzyme mixture comprising 4×CFLP buffer,pH 8.2, 0.8 mM MnCl₂ (to yield a final concentration of 1×CFLP bufferand 0.2 mM MnCl₂) and 0.5 μl of CLEAVASE BN enzyme (50 ng/μl in 1×dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50 mMKCl, 10 μg/ml BSA)). A 20 μl no salt, no enzyme double-strandedsubstrate control was set up in parallel; this reaction containedsterile distilled water in place of the CLEAVASE BN enzyme.

After 5 minutes at 65° C., the reactions were stopped by the addition of16 μl of stop buffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue,0.05% xylene cyanol). The samples were then heated to 72° C. for 2minutes and the reaction products were resolved by electrophoresisthrough a 10% polyacrylamide gel (19:1 cross-link), with 7M urea, in abuffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, as describedin Example 10a.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in example 10a. The DNA was transferred tothe membrane and the membrane was dried, blocked in 1× I-BLOCK (Tropix,Bedford, Mass.), conjugated with streptavidin-alkaline phosphatase(United States Biochemical), washed, reacted with CDPSTAR reagent(Tropix, Bedford, Mass.), and exposed to X-ray film as described inExample 22a. The resulting autoradiograph is shown in FIG. 56.

In FIG. 56, lanes 1-3 contain reaction products derived from reactionscontaining the single-stranded substrate; lanes 4-7 contain reactionproducts derived from reactions containing the double-strandedsubstrate. Lanes land 3 contain 7.0 μl of the reaction products derivedfrom the cleavage reactions which contained either 50 or 100 fmoles ofthe single-stranded substrate, respectively. Lane 2 contains 7.0 μl ofthe uncut single-stranded substrate control reaction. Lanes 4 and 6contain 7.0 μl of the uncut double-stranded control reactions whichcontained either 33 or 66 fmoles of the substrate, respectively. Lanes 5and 7 contain 7.0 μl of the reaction products derived from cleavagereactions which contained either 33 or 66 fmoles of the double-strandedsubstrate, respectively. Note that the uncut double-stranded controlshows a doublet underneath the prominent band containing the 157 bysubstrate; it is believed that this doublet represents alternativestructures which migrate with an altered mobility rather thandegradation products. This doublet does not appear in experimentsperformed using double-stranded DNA purified from a denaturing gel (SeeExample 24).

Comparison of the cleavage patterns generated by cleavage of either thesingle-stranded or double-stranded substrate shows that identicalpatterns are generated.

Example 24 The CLEAVASE Enzyme Reaction Using a Double Stranded DNATemplate is Sensitive to Large Changes in Reaction Conditions

The results presented in Example 13 demonstrated that the CLEAVASEenzyme reaction is relatively insensitive to significant changes innumerous reaction conditions including, the concentration of MnCl₂ andKCl, temperature, the incubation period, the amount of CLEAVASE BNenzyme added and DNA preparation. The results shown in Example 13demonstrated that when the CLEAVASE enzyme reaction is performed using asingle-stranded substrate, the reaction is remarkably robust to largechanges in conditions. The experiments shown below show that thecleavage of double-stranded substrates is restricted to a somewhatnarrower range of reaction conditions.

a) Generation of the Double-Stranded 157 by Fragment of Exon 40f theTyrosinase Gene

The following experiments examine the effect of changes in reactionconditions when double-stranded DNA templates are used in the CLEAVASEenzyme reaction. The double-stranded substrate utilized was the 157 byfragment of the wild type tyrosinase gene (SEQ ID NO:40). This 157 byfragment was generated using symmetric

PCR as described in Example 10b. Briefly, approximately 75 fmoles ofdouble-stranded substrate DNA were incubated with 50 pmoles of theprimer 5′ biotin-GCCTTATTTTACTTTAAAAAT-3′ (SEQ ID NO: 45), 50 pmoles ofthe primer 5′ fluorescein-TAAAGTTTTGTGTTATCTCA-3′ (SEQ ID NO:46), 50 mMof each dNTP, 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05%TWEEN 20 and 0.05% NONIDET P-40 (NP40). Tubes containing 95 μl of theabove mixture were heated to 95° C. for 5 seconds and cooled to 70° C.Five microliters of enzyme mix containing 1.25 units of Taq DNApolymerase in 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05%TWEEN 20 and 0.05% NONIDET P-40 were then added. Each tube was overlaidwith 50 μl of Chill Out 14™ (MJ Research, Watertown, Mass.).

The tubes were heated to 95° C. for 45 seconds, cooled to 50° C. for 45seconds, heated to 72° C. for 75 seconds for 30 repetitions with a 5minute incubation at 72° C. after the last repetition. The reactionswere then ethanol precipitated to reduce the volume to be gel purified.NaCl was added to a final concentration of 400 mM and glycogen (indistilled water) was added to a final concentration of 200 μg/ml. Twoand one-half volumes of 100% ethanol were added to each tube, and thetubes were chilled to −70° C. for two and one-half hours. The DNA waspelleted and resuspended in one-fifth the original volume of steriledistilled water.

The PCR products were gel purified as follows. An equal volume of stopbuffer (95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol) wasadded to each tube and the tubes were heated to 72° C. for 2 minutes.The products were resolved by electrophoresis through a 6% denaturingpolyacrylamide gel (19:1 cross-link) and 7 M urea in a buffer containing45 mM Tris-Borate, pH 8.3 and 1.4 mM EDTA. The DNA was visualized byethidium bromide staining and the 157 by fragment was excised from thegel. The DNA was eluted from the gel slice by passive diffusion asdescribed in Example 10a with the exception that diffusion was allowedto occur over two days at room temperature. The DNA was thenprecipitated with ethanol in the presence of 200 mM NaCl and no addedcarrier molecules. The DNA was pelleted and resuspended in 150 μl TE (10mM Tris-Cl, pH 8.0, 0.1 mM EDTA).

b) Effect of KCl Concentration on the Double-Stranded Cleavage Reaction

To determine the effect of salt concentration upon the cleavage reactionwhen a double-stranded substrate was utilized, a single substrate wasincubated in the presence of a fixed amount of the enzyme CLEAVASE BNenzyme (25 ng) in a buffer containing 10 mM MOPS, pH 7.5, 0.2 mM MnCl₂and varying concentrations of KCl from 0 to 100 mM.

Approximately 100 fmoles of the 157 by fragment derived from the exon 4of the tyrosinase gene (SEQ ID NO:40; prepared as described above insection a) were placed in 200 μl thin wall microcentrifuge tubes(BioRad, Richmond, Calif.) in sterile distilled water in a volume of6.25 μl (the final reaction volume was 10 μl). The tubes were heated to95° C. for 15 seconds and then rapidly cooled to 45° C. The cleavagereactions were started by the addition of 3.75 μl of an enzyme mixcontaining 2.7×CFLP buffer, pH 7.5 (to yield a final concentration of1×), 0.53 mM MnCl₂ (to yield a final concentration of 0.2 mM), 0.5 μlCLEAVASE BN enzyme (50 ng/μl in 1× dilution buffer (0.5% NP40, 0.5%TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg/ml BSA)), and KCl toyield a final concentration of 0, 2.5, 5, 10, 15, 20, 25, 30, 50 or 100mM. The final reaction volume was 10 μl. The enzyme solution was broughtto room temperature before addition to the cleavage reaction. No enzyme(i.e., uncut) controls were set up in parallel at either 0 or 100 mMKCl, with the difference that sterile distilled water was substitutedfor the CLEAVASE BN enzyme.

After 5 minutes at 45° C., the reactions were stopped by the addition of8 μl of stop buffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue,0.05% xylene cyanol). Samples were heated to 72° C. for 2 minutes and 4μl of each reaction were resolved by electrophoresis through a 10%polyacrylamide gel (19:1 cross-link), with 7 M urea, in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1× I-BLOCK BlockingBuffer, washed and exposed to X-ray film as described in Example 22a,except that the distilled water washes were omitted. The resultingautoradiograph is shown in FIG. 57.

In FIG. 57, the lane marked “M” contains molecular weight markers. Lane1 contains the uncut control in 0 mM KCl and shows the mobility of theuncleaved template DNA. Lanes 2 through 11 contain reaction productsgenerated by incubation of the substrate in the presence of CLEAVASE BNenzyme in a buffer containing 0, 2.5, 5, 10, 15, 20, 25, 30, 50, or 100mM KCl, respectively. Lane 12 contains the uncut control incubated in abuffer containing 100 mM KCl.

The results shown in FIG. 57 demonstrate that the CLEAVASE enzymereaction carried out on double-stranded DNA template was sensitive tovariations in salt concentration. Essentially no cleavage was detectedin reactions containing greater than 30 mM KCl. The same cleavagepattern was obtained when the 157 by tyrosinase DNA substrate (SEQ IDNO:40) was incubated with the CLEAVASE BN enzyme regardless of whetherthe concentration of KCl was varied from 0 to 30 mM.

c) Effect of NaCl on the Double-Stranded Cleavage Reaction

The effect of substituting NaCl in place of KCl upon the cleavagepattern created by 5′ nuclease activity on a double-stranded DNAsubstrate was examined. Approximately 100 fmoles of the 157 by fragmentderived from exon 4 of the tyrosinase gene (SEQ ID NO 40; prepared asdescribed in Example 24a) were placed in 200 μl thin wallmicrocentrifuge tubes (BioRad, Richmond, Calif.) in sterile distilledwater in a volume of 6.25 μl and were heated to 95° C. for 15 seconds.The tubes were cooled to 45° C. The cleavage reaction was started by theaddition of 3.75 μl of an enzyme mix containing 2.7×CFLP buffer, pH 7.5(to yield a final concentration of 1×), 0.53 mM MnCl₂ (to yield a finalconcentration of 0.2 mM), 0.5 μl enzyme BN (50 ng/μl in 1× dilutionbuffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10μg/ml BSA)), and NaCl to yield a final concentration of 0, 2.5, 5, 10,15, 20, 25, 30, 50 or 100 mM. The final reaction volume was 10 μl. Noenzyme (i.e., uncut) controls were set up in parallel at either 0 or 100mM NaCl, with the difference that sterile distilled water wassubstituted for the CLEAVASE BN enzyme.

The reactions were incubated at 45° C. for 5 minutes and were stopped bythe addition of 8 μl of stop buffer (95% formamide, 10 mM EDTA, 0.05%bromophenol blue, 0.05% xylene cyanol). Samples were heated to 72° C.for 2 minutes and 4 μl of each reaction were resolved by electrophoresisthrough a 10% polyacrylamide gel (19:1 cross-link), with 7 M urea, in abuffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1× I-BLOCK BlockingBuffer, washed and exposed to X-ray film as described in Example 22awith the exception that the distilled water washes were omitted. Theresulting autoradiograph is shown in FIG. 58.

In FIG. 58, the lane marked “M” contains molecular weight markers. Lane1 contains the no enzyme control incubated in a buffer containing 0 mMNaCl and shows the mobility of the uncleaved template DNA. Lanes 2through 11 contain reaction products generated by cleavage of the 157 bysubstrate (SEQ ID NO:40) with the CLEAVASE BN enzyme in a buffercontaining 0, 2.5, 5, 10, 15, 20, 25, 30, 50, or 100 mM NaCl,respectively. Lane 12 contains the no enzyme control incubated in abuffer containing 100 mM NaCl.

The results shown in FIG. 58 demonstrate that the CLEAVASE enzymereaction carried out on a double-stranded DNA template was sensitive tovariations in NaCl concentration. Essentially no cleavage was detectedabove 20 mM NaCl. The same cleavage pattern was obtained when the 157 bytyrosinase DNA template (SEQ ID NO:40) was incubated with the CLEAVASEBN enzyme regardless of whether the NaCl concentration was varied from 0to 20 mM.

d) Effect of Substituting (NH₄)₂SO₄ for KCl in Cleavage ofDouble-Stranded Template

In an approach similar to that described in Example 22b, the ability of(NH₄)₂SO₄ to substitute for KCl in the cleavage reaction whendouble-stranded substrates were utilized was tested. Cleavage reactionswere set up exactly as described in Examples 24b and c with theexception that variable amounts of (NH₄)₂SO₄ were substituted for theKCl or NaCl.

Approximately 100 fmoles of the 157 by fragment derived exon 4 of thetyrosinase gene (SEQ ID NO 40; prepared as described above in section a)were placed in 200 μl thin wall microcentrifuge tubes (BioRad, Richmond,Calif.) in sterile distilled water in a volume of 6.25 μl and wereheated to 95° C. for 15 seconds. The tubes were cooled to 45° C.

Cleavage reactions were started by the addition of 3.75 μl of an enzymemix containing 2.7×CFLP buffer, pH 7.5 (to yield a final concentrationof 1×), 0.53 mM MnCl₂ (to yield a final concentration of 0.2 mM MnCl₂),0.5 μl CLEAVASE BN enzyme (50 ng/μl in 1× dilution buffer (0.5% NP40,0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg/ml BSA)), and(NH₄)₂SO₄ to yield a final concentration of 0, 2.5, 5, 10, 15, 20, 25,30, 50 or 100 mM. The final reaction volume was 10 μl. No enzyme (i.e.,uncut) controls were set up in parallel at either 0 or 100 mM (NH₄)₂SO₄,with the difference that sterile distilled water was substituted for theCLEAVASE BN enzyme.

The reactions were incubated at 45° C. for 5 minutes and were stopped bythe addition of 8 μl of stop buffer (95% formamide, 10 mM EDTA, 0.05%bromophenol blue, 0.05% xylene cyanol). Samples were heated to 72° C.for 2 minutes and 4 μl of each reaction were resolved by electrophoresisthrough a 10% polyacrylamide gel (19:1 cross-link), with 7 M urea, in abuffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1× I-BLOCK BlockingBuffer, washed and exposed to X-ray film as described in Example 22a,except that the distilled water washes were omitted. The resultingautoradiograph is shown in FIG. 59.

In FIG. 59, the lane marked “M” contains molecular weight markers. Lane1 contains the no enzyme control incubated in a buffer containing 0 mM(NH₄)₂SO₄ and shows the migration of the uncleaved substrate DNA. Lanes2 through 11 contain reaction products generated by incubation of thesubstrate in the presence of CLEAVASE BN enzyme in a buffer containing0, 2.5, 5, 10, 15, 20, 25, 30, 50, or 100 mM (NH₄)₂SO₄, respectively.Lane 12 contains the no enzyme control incubated in a buffer containing100 mM (NH₄)₂SO₄.

The results shown in FIG. 59 demonstrate that the CLEAVASE enzymereaction was severely inhibited by the presence of (NH₄)₂SO₄. Thereaction was completely inhibited by as little as 15 mM (NH₄)₂SO₄; theextent of the cleavage reaction in 5 mM (NH₄)₂SO₄ was comparable to thatobtained in 20 mM KCl and was significantly reduced relative to thatobtained in 0 mM (NH₄)₂SO₄. The pattern of cleavage obtained using 5 mM(NH₄)₂SO₄, however, was identical to that observed when the 157 bysubstrate was incubated in the absence of (NH₄)₂SO₄ or in KCl or NaCl,indicating that the choice of salt included in the CLEAVASE enzymereaction has no effect on the nature of the sites recognized by theenzyme.

e) Time Course of the Double-Stranded Cleavage Reaction

To determine how quickly the double-stranded cleavage reaction iscompleted, a single substrate was incubated in the presence of a fixedamount of CLEAVASE BN enzyme for various lengths of time. Approximately100 fmoles of the double-stranded 157 by fragment of exon 4 of thetyrosinase gene (SEQ ID NO 40; prepared as described above in Example24a) were placed in sterile distilled water in 200 μl thin walledcentrifuge tubes (BioRad, Richmond, Calif.) in a volume of 6.25 μl. Thetubes were heated to 95° C. for 15 seconds, as described in section b),and cooled to 45° C.

Cleavage reactions were started by the addition of 3.75 μl of an enzymemix containing 2.7×CFLP buffer, pH 7.5 (to yield a final concentrationof 1×), 0.53 mM MnCl₂ (to yield a final concentration of 0.2 mM MnCl₂),0.5 μl CLEAVASE BN enzyme (50 ng/μl in 1× dilution buffer (0.5% NP40,0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg/ml BSA)). Thefinal reaction volume was 10 μl. No enzyme (i.e., uncut) controls wereset up in parallel and stopped after either 5 minutes or 120 minutes,with the difference that sterile distilled water was substituted for theCLEAVASE BN enzyme.

The cleavage reactions were stopped by the addition of 8 μl of stopbuffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue, 0.05% xylenecyanol) at the following times: 5 seconds, 1, 2, 5, 10, 15, 20, 30, 60or 120 minutes. Samples were heated to 72° C. for 2 minutes and 4 μl ofeach reaction were resolved by electrophoresis through a 10%polyacrylamide gel (19:1 cross-link), with 7 M urea, in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1× I-BLOCK BlockingBuffer, washed and exposed to X-ray film as described in Example 22awith the exception that the distilled water washes were omitted. Theresulting autoradiograph is shown in FIG. 60.

In FIG. 60, lane 1 contains the no enzyme control after a 5 minuteincubation at 45° C. and shows the mobility of the uncleaved templateDNA. Lanes 2-10 contain cleavage fragments derived from reactionsincubated in the presence of CLEAVASE BN for 5 sec, 1, 2, 5, 10, 15, 20,30, 60 (i hr), or 120 minutes (2 hr), respectively. Lane 11 contains theno enzyme control after a 120 minute incubation at 45° C.

FIG. 60 shows that the cleavage of a double-stranded DNA templatemediated by the CLEAVASE BN enzyme was rapid. A full cleavage patternwas apparent and essentially complete within one minute. Unlike theexample of cleavage of a single-stranded DNA template (Example 13c),very little cleavage is detectable after 5 seconds. This reactioncontained one-tenth the amount of enzyme used in the reaction describedin Example 13c. In addition, whereas incubation of single-strandedcleavage reactions for extended periods generated a pattern ofincreasingly truncated fragments (Example 22e), extended incubation ofthe double-stranded cleavage reaction resulted in a complete loss ofsignal (FIG. 60, lane 10); this result is probably due to nibbling bythe enzyme of the 5′ biotin moiety from the reannealed strands. It isimportant to note that these results show that the same pattern ofcleavage was produced for cleavage of double-stranded DNA, as forsingle-stranded, whether the reaction is run for 1 or 30 minutes. Thatis, the full representation of the cleavage products (i.e., bands) isseen over a 30-fold difference in time of incubation; thus thedouble-stranded CFLP reaction need not be strictly controlled in termsof incubation time.

The results shown in FIG. 61 contain short time courses of cleavagereactions performed at a variety of enzyme concentrations. Approximately100 fmoles of the double-stranded 157 by fragment of exon 4 of thetyrosinase gene (SEQ ID NO:40) were placed in sterile distilled water in200 μl thin walled centrifuge tubes (BioRad, Richmond, Calif.) in avolume of 6.25 μl. The tubes were heated to 95° C. for 15 seconds, asdescribed in Example 24b, and cooled to 45° C. Cleavage reactions werestarted by the addition of 3.75 μl of an enzyme mix containing 2.7×CFLPbuffer, pH 7.5 (to yield a final concentration of 1×), 0.53 mM MnCl₂ (toyield a final concentration of 0.2 mM MnCl₂), 0.5 μl CLEAVASE BN enzyme(at either 50, 100, 200, or 500 ng/μl in 1× dilution buffer (0.5% NP40,0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg/ml BSA) to yielda final amount of enzyme of 25, 50, 100, or 250 ng). The final reactionvolume was 10 μl. A no enzyme control was set up in parallel, with thedifference that sterile distilled water was substituted for the CLEAVASEBN enzyme, and stopped after 1 minute at 45° C.

The cleavage reactions were stopped by the addition of 8 μl of stopbuffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue, 0.05% xylenecyanol) after either 5 seconds or 1 minute. Samples were heated to 72°C. for 2 minutes and 4 μl of each reaction were resolved byelectrophoresis through a 10% polyacrylamide gel (19:1 cross-link), with7 M urea, in a buffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1× I-BLOCK BlockingBuffer, washed and exposed to X-ray film as described in Example 22a,except that the distilled water washes were omitted. The resultingautoradiograph is shown in FIG. 61.

In FIG. 61, lane “M” contains molecular weight markers as described inExample 10a. Lane 1 contains the no enzyme control. Lanes 2 and 3 eachcontain reaction products generated by incubation of the substrate inthe presence of 25 ng of CLEAVASE BN enzyme; the reaction in lane 2 wasstopped after 5 seconds, that in lane 3, after 1 minute. Lanes 4 and 5contain reaction products generated by cleavage of the substrate in thepresence of 50 ng of CLEAVASE BN enzyme; the reaction in lane 4 wasstopped after 5 seconds, that in lane 5, after 1 minute. Lanes 6 and 7contain reaction products generated by cleavage of the substrate in thepresence of 100 ng of CLEAVASE BN enzyme; the reaction in lane 6 wasstopped after 5 seconds, that in lane 7, after 1 minute. The reactionsshown in lanes 8 and 9 each contain 250 ng of CLEAVASE BN enzyme; thatin lane 8 was stopped after 5 seconds, that in lane 9, after 1 minute.

The results presented in FIG. 61 indicate that the rate of cleavage ofdouble-stranded DNA increased with increasing enzyme concentration. Notethat as the concentration of enzyme was increased, there was acorresponding reduction in the amount of uncut DNA that remained after 1minute. As was demonstrated below, in FIG. 63, the concentration ofenzyme included in the cleavage reaction had no effect on the cleavagepattern generated. Comparison of the 250 ng reaction (shown in FIG. 61,lanes 8 and 9) to the short time point digestion described in Example13c, indicates that the amount of enzyme rather than the double-strandedor single-stranded nature of the substrate controls the extent ofcleavage in the very early time points.

f) Temperature Titration of the Double-Stranded Cleavage Reaction

To determine the effect of temperature variation on the cleavagepattern, the 157 by fragment of exon 4 of the tyrosinase gene (SEQ IDNO:40) was incubated in the presence of a fixed amount of CLEAVASE BNenzyme for 5 minutes at various temperatures. Approximately 100 fmolesof substrate DNA (prepared as described in Example 24a) were placed insterile distilled water in 200 μl thin walled centrifuge tubes (BioRad,Richmond, Calif.) in a volume of 6.25 μl. The tubes were heated to 95°C. for 15 seconds and cooled to either 37, 40, 45, 50, 55, 60, 65, 60,75, or 80° C. Cleavage reactions were started by the addition of 3.75 μlof an enzyme mix containing 2.7×CFLP buffer, pH 7.5 (to yield a finalconcentration of 1×), 0.53 mM MnCl₂ (to yield a final concentration of0.2 mM MnCl₂), 0.5 μl CLEAVASE BN enzyme (50 ng/μl in 1× dilution buffer(0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg/mlBSA)). The enzyme mix was kept on ice throughout the duration of theexperiment, but individual aliquots of the enzyme mix were allowed tocome to room temperature before being added to the reactions. A secondreaction was run at 37° C. at the end of the experiment to control forany loss of enzyme activity that may have occurred during the course ofthe experiment. No enzyme controls were set up in parallel and incubatedat either 37° C. or 80° C., with the difference that sterile distilledwater was substituted for the CLEAVASE BN enzyme. The reactions werestopped by the addition of 8 μl of stop buffer (95% formamide, 10 mMEDTA, 0.05% bromophenol blue, 0.05% xylene cyanol).

Samples were heated to 72° C. for 2 minutes and 5 μl of each reactionwere resolved by electrophoresis through a 10% polyacrylamide gel (19:1cross-link), with 7 M urea, in a buffer containing 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1× I-BLOCK BlockingBuffer, washed and exposed to X-ray film as described in Example 22a,except that the distilled water washes were omitted. The resultingautoradiograph is shown in FIG. 62.

In FIG. 62, the lane marked “M” contains molecular weight markers,prepared as described in Example 10a. Lane 1 contains the no enzymecontrol after a 5 minute incubation at 37° C. Lanes 2 and 3 containreactions incubated at 37° C., run at the beginning and end of theexperiment, respectively. Lanes 4-13 contain reactions incubated at 40,45, 50, 55, 60, 65, 70, 75, or 80° C. (there are two 80° C. samples; thefirst was not covered with CHILL OUT 14 (MJ Research, Watertown, Mass.),the second was overlaid with 20 μl CHILL OUT 14 after the addition ofthe enzyme mix), respectively. Lane 14 contains a no enzyme controlincubated at 80° C. for 5 minutes.

FIG. 62 shows that cleavage of double-stranded DNA substrates proceededmost effectively at lower temperatures. The distribution of signal andpattern of cleavage changed smoothly in response to the temperature ofincubation over the range of 37° C. to 60° C. Some cleavage productswere evident only upon incubation at higher temperatures, whereas otherswere far more predominant at lower temperatures. Presumably, certainstructures that are substrates for the CLEAVASE BN enzyme at one end ofthe temperature range are not favored at the other. As expected, theproduction of cleavage fragments became progressively less abundant inthe high end of the temperature range as the cleavage structures weremelted out. Above 70° C., the cleavage products were restricted to smallfragments, presumably due to extensive denaturation of the substrate.When longer DNAs (350 to 1000 nucleotides) are used, it has been foundthat useful patterns of cleavage are generated up to 75° C.

These results show that the cleavage reaction can be performed over afairly wide range of temperatures using a double-stranded DNA substrate.As in the case of the single-stranded cleavage reaction, the ability tocleave double-stranded DNA over a range of temperatures is important.Strong secondary structures that may dominate the cleavage pattern arenot likely to be destabilized by single-base changes and may thereforeinterfere with mutation detection. Elevated temperatures can then beused to bring these persistent structures to the brink of instability,so that the effects of small changes in sequence are maximized andrevealed as alterations in the cleavage pattern. This also demonstratesthat within the useful temperature range, small changes in the reactiontemperature due to heating block drift or similar device variations willnot cause radical changes in the cleavage pattern.

g) Titration of the CLEAVASE BN Enzyme in Double-Stranded CleavageReactions

The effect of varying the concentration of the CLEAVASE BN enzyme in thedouble-stranded cleavage reaction was examined. Approximately 100 fmolesof the 157 by fragment of exon 4 of the tyrosinase gene (SEQ ID NO:40;prepared as described in Example 24a) were placed in sterile distilledwater in 200 μl thin walled centrifuge tubes (BioRad, Richmond, Calif.)in a total volume of 6.25 μl. These tubes were heated to 95° C. for 15seconds and then rapidly cooled to 45° C.

Cleavage reactions were started immediately by the addition of 3.75 μlof a diluted enzyme mix containing 2.7×CFLP buffer, pH 7.5 (to yield afinal concentration of 1×), 0.53 mM MnCl₂ (to yield a finalconcentration of 0.2 mM MnCl₂), 0.5 μl CLEAVASE BN enzyme (2, 10, 20,50, 100, 200, 500 ng/μl in 1× dilution buffer (0.5% NP40, 0.5% TWEEN 20,20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg/ml BSA) such that 1, 5, 10, 25,50, 100 or 250 ng of enzyme was added to the reactions). No enzymecontrols were set up in parallel, with the difference that 1× dilutionbuffer was substituted for the CLEAVASE BN enzyme. After 5 minutes at45° C., the reactions were stopped by the addition of 8 μl of stopbuffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue, 0.05% xylenecyanol). The samples were heated to 72° C. for 2 minutes and 4 μl ofeach reaction were resolved by electrophoresis through a 10%polyacrylamide gel (19:1 cross-link), with 7 M urea, in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1× I-BLOCK BlockingBuffer, washed and exposed to X-ray film as described in Example 22a,except that the distilled water washes were omitted. The resultingautoradiograph is shown in FIG. 63.

The lane marked “M” in FIG. 63 contains molecular weight markers. Lane 1contains the no enzyme control and shows the migration of the uncutsubstrate. Lanes 2-8 contain cleavage products derived from reactionscontaining 1, 5, 10, 25, 50, 100 or 250 ng of the CLEAVASE BN enzyme,respectively.

These results show that the same cleavage pattern was obtained using the157 by tyrosinase DNA substrate (SEQ ID NO:40) regardless of whether theamount of enzyme used in the reaction varied over a 50-fold range. Thus,the double-stranded cleavage reaction is ideally suited for practice inclinical laboratories where reaction conditions are not as controlled asin research laboratories. Note, however, that there is a distinctoptimum for cleavage at intermediate enzyme concentrations for adouble-stranded template, in marked contrast to what was observed onsingle-stranded substrates (Example 13e). The progressive loss of signalin the double-stranded reactions at increasing concentrations ofCLEAVASE BN enzyme is likely due to the nibbling of the 5′ biotin labeloff the end of the reannealed double-stranded template.

Example 25 Determination of the pH Optimum for Single-Stranded andDouble-Stranded Cleavage Reactions

In order to establish optimal pH conditions for the two types ofprimer-independent cleavage reactions (i.e., single-stranded anddouble-stranded cleavage reactions), the CLEAVASE enzyme reaction bufferwas prepared at a range of different pHs.

a) Establishment of a pH Optimum for the Single-Stranded CleavageReaction

The effect of varying the pH of the CLEAVASE enzyme reaction (i.e., CFLPreaction) buffer upon the cleavage of single-stranded substrates wasexamined. Several 10× buffer solutions were made with 0.5 M MOPS at pH6.3, 7.2, 7.5, 7.8, 8.0 and 8.2 by titrating a 1 M solution of MOPS atpH 6.3 with 6 N NaOH. The volume was then adjusted to yield a 0.5 Msolution at each pH.

Approximately 100 fmoles of a single-stranded substrate prepared fromthe sense strand of exon 4 of the tyrosinase gene (SEQ ID NO:47;prepared as described in Example 10a), were placed in 200 μl thin walledcentrifuge tubes (BioRad, Richmond, Calif.) in 15 μl of 1×CFLP buffer,at varying pH, and 1.33 mM MnCl₂ (to yield a final concentration of 1mM). The final reaction volume was 20 μl. The reaction mixes were heatedto 95° C. for 5 seconds and rapidly cooled to 65° C. The reactions werestarted by the addition of 5 μl of diluted enzyme mix containing 1 μl ofCLEAVASE BN enzyme (50 ng/μl in 1× dilution buffer (0.5% NP40, 0.5%TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg/ml BSA)) in 1×CFLPbuffer (without MnCl₂), again at the appropriate pH. A 20 μl no salt, noenzyme control was set up in parallel and incubated at 65° C. for eachof the indicated pHs, with the difference that sterile distilled waterwas substituted for CLEAVASE BN enzyme and all reaction components wereadded prior to denaturation. Reactions were stopped by the addition of16 μl of stop buffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue,0.05% xylene cyanol) after 5 minutes.

Samples were heated to 72° C. for 2 minutes and 7 μl of each reactionwere resolved by electrophoresis through a 10% polyacrylamide gel (19:1cross-link), with 7M urea, in a buffer containing 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA, as described in Example 10a.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in Example 10a. The DNA was transferred tothe membrane and the membrane was dried, blocked in 1× I-BLOCK (Tropix,Bedford, Mass.), conjugated with streptavidin-alkaline phosphatase(United States Biochemical), washed, reacted with CDPSTAR reagent(Tropix, Bedford, Mass.), and exposed to X-ray film as described inExample 22a, except that the distilled water washes were omitted. Theresults are presented in FIG. 64.

In FIG. 64, panels A and B contain reactions which used single-strandedDNA substrates. In panel A, 5 pairs of reactions are presented. In eachcase, the first lane of the pair is the no enzyme control and the secondis the single-stranded cleavage reaction. Lanes 1 and 2 depict reactionproducts obtained using a reaction buffer at pH 6.3; lanes 3 and 4, atpH 7.2; lanes 5 and 6, pH 7.8; lanes 7 and 8, pH 8.0; lanes 9 and 10, atpH 8.2. Panel B contains the results of a separate experiment comparingcleavage reactions performed using a reaction buffer at pH 7.5 (lanes 1and 2, uncut and cut, respectively) and at pH 7.8 (lanes 3 and 4, uncutand cut, respectively).

The results shown in FIG. 64, panels A and B, indicate that the cleavageof the single-stranded DNA template was sensitive to relatively smallchanges in pH. There was a pH optimum for the reaction between pH 7.5and 8.0. Because the pK_(a) of MOPS is 7.2, the pH closest to that valuewhich supported cleavage, pH 7.5, was determined to be optimal for thesingle-stranded cleavage reaction.

b) Establishment of a pH Optimum for the Double-Stranded CleavageReaction

The effect of varying the pH of the CLEAVASE enzyme reaction (i.e., CFLPreaction) buffer upon the cleavage of double-stranded substrates wasexamined. Several 10× buffer solutions were made with 0.5 M MOPS at pH7.2, 7.5, 7.8, and 8.0, as described above in section a). Approximately100 fmoles of the double-stranded 157 by fragment of exon 4 of thetyrosinase gene (SEQ ID NO:40; prepared as described in Example 10) wereplaced in 200 μl thin walled centrifuge tubes (BioRad, Richmond, Calif.)in a total volume of 6.25 μl. The tubes were heated to 95° C. for 15seconds and cooled to 45° C. The clevage reactions were started by theaddition of 3.75 μl of diluted enzyme mix containing 2.7×CFLP buffer, pH7.5 (to yield a final concentration of 1×), 0.53 mM MnCl₂ (to yield afinal concentration of 0.2 mM MnCl₂), 0.5 μl of CLEAVASE BN enzyme (50ng/μl in 1× dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl,pH 8.0, 50 mM KCl, 10 μg/ml BSA)).

The cleavage reactions were incubated for 5 minutes and then wereterminated by the addition of 8 μl of stop buffer (95% formamide, 10 mMEDTA, 0.05% bromophenol blue, 0.05% xylene cyanol).

Samples were heated to 72° C. for 2 minutes and 4 μl of each reactionwere resolved by electrophoresis through a 10% polyacrylamide gel (19:1cross-link), with 7M urea, in a buffer containing 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in Example 10b. The DNA was transferred tothe membrane and the membrane was dried, washed in 1× I-BLOCK BlockingBuffer, washed and exposed to X-ray film as described in Example 22a,except that the distilled water washes were omitted. The resultingautoradiographs are shown in FIG. 65, panels A and B.

In FIG. 65, panel A, lanes 1 and 2 contain cleavage products fromreactions run in a buffer at pH 8.2 (lane 1 contains the cleavagereaction; lane 2 is the uncut control). Lanes 3 and 4 contain cleavageproducts from reactions run in a buffer at pH 7.2 (lane 3 contains thecleavage reaction; lane 4 is the uncut control). In panel B, lanes 1 and2 contain cleavage products from reactions run in a buffer at pH 7.5(lane 1 is the uncut control; lane 2 contains the cleavage reaction).Lanes 3 and 4 contain cleavage products from reactions run in a bufferat pH 7.8 (lane 3 contains the uncut control; lane 4 contains thecleavage reaction).

The results in FIG. 65, panels A and B, demonstrate that the cleavage ofdouble-stranded DNA was not sensitive to changes in pH over the range ofbuffer conditions tested. Because the cleavage of single-stranded DNA,however, was sensitive to changes in pH, the buffer conditions that weredetermined to be optimal for the single-stranded cleavage reaction werechosen for subsequent double-stranded cleavage experiments.

Example 26 The Presence of Competitor DNA does not Alter the CleavagePattern

The effect of the presence of competitor (i.e., non-labelled substrate)DNA upon the cleavage reaction was examined. The cleavage reaction wasrun using the 157 nucleotide fragment from the sense strand of the humantyrosinase gene (SEQ ID NO:47) and human genomic DNA. The results shownbelow demonstrate that the presence of non-substrate DNA has no effecton the CFLP pattern obtained in the cleavage reaction.

a) Preparation of the Substrate DNA and the Cleavage Reactions

The 157 nucleotide single-stranded wild type tyrosinase substrate (SEQID NO:47) containing a biotin label on the 5′ end was prepared asdescribed in Example 11. Human genomic DNA (Promega) present at 235μg/ml in Tris-HCl, pH 8.0; 1 mM EDTA was ethanol precipitated andresuspended in Tris-HCl, pH 8.0; 0.1 mM EDTA to final concentration 400μg/ml. This DNA was used as a competitor in standard CFLPsingle-stranded reactions (described in Example 11). Tyrosinase DNAsubstrate (SEQ ID NO:47) and human genomic DNA were mixed in H₂O infinal volume of 6 μl. Samples were heated at 95° C. for 10 seconds todenature the DNA, cooled to the target temperature of 65° C., andmixture of 2 μl 5×CFLP buffer, pH 7.5, 1 μl 10 mM MnCl₂ and 1 μl (25 ng)the enzyme CLEAVASE BN in dilution buffer was added. After 5 minutes at65° C., 6 μl of stop buffer was added to terminate reaction and 5 μl ofeach sample was separated on a 10% denaturing polyacrylamide gel.Membrane transfer and DNA visualization were performed as described inExample 21.

b) The Presence of Genomic DNA does not Alter the CFLP Pattern

FIG. 66 shows the resulting pattern corresponding to the cleavageproducts of the sense strand of the wild type tyrosinase substrate (SEQID NO:47) in the presence of 0 μg/ml (lane 2), 20 μg/ml (lane 3), 40μg/ml (lane 4), 80 μg/ml (lane 5), 120 μg/ml (lane 6) and 200 μg/ml(lane 7) unlabeled human genomic DNA. Lane 1 shows an uncut control inthe absence of the enzyme CLEAVASE BN and lane marked “M” contains themolecular weight markers prepared as described in Example 10.

FIG. 66 shows that the presence of genomic DNA in the cleavage reactiondid not change either the position or the relative intensity of theproduct bands produced. Increasing the amount of nonspecific DNA in thereaction did, however, decrease the efficiency of the cleavage reactionand reduced the overall intensity of the pattern. These results can beexplained by the binding of the CLEAVASE BN enzyme to the nonspecificDNA which has the effect of decreasing the effective enzymeconcentration in the reaction. This effect became significant when theconcentration of genomic DNA in the reaction was equal to or greaterthan 120 μg/ml (FIG. 66, lanes 6 (120 μg/ml) and 7 (200 μg/ml)). Underthese conditions, the genomic DNA was present at more than a 20.000-foldexcess relative to the specific substrate DNA; nonetheless the CFLPpattern could still be recognized under these conditions. The observedstability of the CFLP pattern in the presence of genomic DNA ruled outthe possibility that nonspecific DNA could significantly change thestructure of the substrate DNA or alter the interaction of the CLEAVASEBN enzyme with the substrate.

Example 27 The CFLP Reaction can be Practiced Using a Variety of Enzymes

The above Examples demonstrated the ability of CLEAVASE BN enzyme, a 5′nuclease derived from Taq DNA polymerase, to generate a characteristicset of cleavage fragments from a nucleic acid substrate. The followingexperiments demonstrate that a number of other enzymes can be used togenerate a set of cleavage products which are characteristic of a givennucleic acid. These enzymes are not limited to the class of enzymescharacterized as 5′ nucleases.

a) Cleavage Patterns Generated by Other DNA Polymerases from the GenusThermus

To determine whether 5′ nuclease activity associated with DNApolymerases (DNAPs) other than Taq DNAP could generate a distinctcleavage pattern from nucleic acid substrates, DNAPs from two species ofThermus were examined. The DNAP of Thermus flavus (“Tfl”, Kaledin etal., Biokhimiya 46:1576 (1981); obtained from Promega Corp., Madison,Wis.) and the DNAP of Thermus thermophilus (“Tth”, Carballeira et al.,Biotechniques 9:276 (1990); Myers et al., Biochem. 30:7661 (1991);obtained from U.S. Biochemicals, Cleveland, Ohio) were examined fortheir ability to generate suitable cleavage patterns (i.e., patternswhich can be used to characterize a given nucleic acid substrate).

The ability of these other enzymes to cleave nucleic acids in astructure-specific manner was tested using the single-stranded 157nucleotide fragment of the sense strand of exon 4 of the tyrosinase gene(SEQ ID NO:47) under conditions reported to be optimal for the synthesisof DNA by each enzyme.

Approximately 100 fmoles of the 157 nucleotide fragment derived from thesense strand of exon 4 of the tyrosinase gene (SEQ ID NO 47; prepared asdescribed in example 10a) were placed in 200 μl thin wallmicrocentrifuge tubes (BioRad, Richmond, Calif.) in 1×CFLP buffer, pH8.2 and 1.33 mM MnCl₂ (to yield a final concentration of 1 mM) and KClto yield a final concentration of either 0 or 50 mM. Final reactionvolumes were 20 μl. Samples were heated to 95° C. for 5 seconds and thencooled to 65° C. A 20 μl no salt, no enzyme control was set up inparallel, with the differences that sterile distilled water wassubstituted for CLEAVASE BN enzyme and all reaction components wereadded prior to denaturation at 95° C.

The cleavage reactions were started by the addition of 5 μl of a dilutedenzyme mix containing either 1.25 units or 5 units of the indicatedenzyme (see below) in 1×CFLP buffer, pH 8.2. After 5 minutes, reactionswere stopped by the addition of 16 μl of stop buffer (95% formamide, 10mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol).

Samples were heated to 72° C. for 2 minutes and 7 μl (in the case of thesamples digested with Tfl) or 5 μl (in the case of the samples digestedwith Tth) were electrophoresed through a 10% polyacrylamide gel (19:1cross-link), with 7M urea, in a buffer containing 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA, as described in Example 10a.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in Example 10a. The DNA was transferred tothe membrane and the membrane was dried, blocked in 1× I-BLOCK (Tropix,Bedford, Mass.), conjugated with streptavidin-alkaline phosphatase(United States Biochemical, Cleveland, Ohio), washed, reacted withCDPSTAR reagent (Tropix, Bedford, Mass.), and exposed to X-ray film asdescribed in Example 22a, except that the distilled water washes wereomitted. The results are presented in FIGS. 67 and 68.

In FIG. 67, lane 1 contains the no enzyme control and indicates themigration of the uncut DNA. Lanes 2-5 contain cleavage products derivedfrom reactions incubated with Tfl DNAP. The reactions represented inlane 2 and 3 each contained 5 units of Tfl DNAP; the sample in lane 2was incubated in a reaction buffer containing 0 mM KCl, while the samplein lane 3 was incubated in a reaction buffer containing 50 mM KCl. Thereactions in lanes 4 and 5 each contained 1.25 units of Tfl DNAP; thesample in lane 4 was incubated in a reaction buffer containing 0 mM KCl;that in lane 5 was incubated in a reaction buffer containing 50 mM KCl.

In FIG. 68, lanes 1 and 2 each contain cleavage products derived fromreactions incubated with 1.25 units of Tth DNAP. The sample in lane 1was incubated in a reaction buffer containing 0 mM KCl; that in lane 2was incubated in a reaction buffer containing 50 mM KCl. Lanes 3 and 4contain cleavage products derived from reactions incubated with 5 unitsof Tth DNAP. The sample shown in lane 3 was incubated in a reactionbuffer containing 0 mM KCl; that in lane 4 was incubated in a reactionbuffer containing 50 mM KCl.

FIGS. 67 and 68 demonstrates that both Tth DNAP and Tfl DNAP displaystructure specific endonuclease activity similar in nature to that seenin the CLEAVASE BN enzyme. A comparison of the results shown in FIGS. 67and 68 showed that the Tth DNAP was more efficient at generating acleavage pattern under the reaction conditions tested. Comparison of thecleavage patterns generated by Tth DNAP with those generated by theCLEAVASE BN enzyme the indicates that essentially the same structuresare recognized by these two enzymes (compare FIG. 69, lane 2 (CLEAVASEBN enzyme) with FIG. 68 (Tth DNAP)).

b) Enzymes Characterized as 3′ Nucleases Can be Used to GenerateDistinct Clevage Patterns

To determine whether enzymes possessing 3′ nucleolytic activity couldalso generate a distinct cleavage pattern, enzymes other than DNAPs(which possess 5′ nuclease activity) were tested in the cleavagereaction. Exonuclease III from Escherichia coli (E. coli Exo III) wastested in a cleavage reaction using the 157 nucleotide fragment preparedfrom the sense strand of exon 4 of the tyrosinase gen (SEQ ID NO:47). Asa comparison, a reaction containing this substrate (SEQ ID NO:47) andCLEAVASE BN enzyme was also prepared.

Approximately 100 fmoles of the 157 nucleotide fragment prepared fromthe sense strand of exon 4 of the tyrosinase gene (SEQ ID NO:47;prepared as described in Example 10a) were placed in 200 μl thin wallmicrocentrifuge tubes (BioRad, Richmond, Calif.) in 1×CFLP buffer, pH8.2 and 1.33 mM MnCl₂ (to yield a final concentration of 1 mM) and KClto yield a final concentration of either 0 or 50 mM in a volume of 15μl. Final reaction volumes were 20 μl.

The samples were heated to 95° C. for 5 seconds and then rapidly cooledto 37° C. A 20 μl no salt, no enzyme control was set up in parallel,with the differences that sterile distilled water was substituted forCLEAVASE BN enzyme and all reaction components were added prior todenaturation at 95° C.

A reaction tube containing 100 fmoles of the 157 nucleotide fragment(SEQ ID NO:47) and 50 ng of CLEAVASE BN enzyme in a buffer containing 0mM KCl was prepared and treated as described in Example 23 (i.e.,denatured by incubation at 95° C. for 5 seconds followed by cooling to65° C. and the addition of the enzyme and incubation at 65° C. for 5minutes).

The cleavage reactions were started by the addition of 5 μl of a dilutedenzyme mix containing either 1.25 units or 200 units of Exo III (UnitedStates Biochemical, Cleveland, Ohio) in 1×CFLP buffer, pH 8.2 (withoutMnCl₂) were added to the 15 μl reactions, and the reactions wereincubated for 5 minutes. After 5 minutes at 37° C., the reactions werestopped by the addition of 16 μl of stop buffer (95% formamide, 10 mMEDTA, 0.05% bromophenol blue, 0.05% xylene cyanol).

The samples were heated to 72° C. for 2 minutes and 5 μl wereelectrophoresed through a 10% polyacrylamide gel (19:1 cross-link), with7M urea, in a buffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA,as described in Example 10a.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in example 10a. The DNA was transferred tothe membrane and the membrane was dried, blocked in 1× I-BLOCK (Tropix,Bedford, Mass.), conjugated with streptavidin-alkaline phosphatase(United States Biochemical, Cleveland, Ohio), washed, reacted withCDPSTAR reagent (Tropix, Bedford, Mass.), and exposed to X-ray film asdescribed in Example 22a, except that the distilled water washes wereomitted. The results are presented in FIG. 69.

Lane 1 in FIG. 69 contains the no enzyme control and indicates themobility of the uncut DNA. Lane 2 contains cleavage fragments generatedby incubation of the substrate with CLEAVASE BN enzyme and provides acomparison of the patterns generated by the two different enzymes. Lanes3-6 contain cleavage fragments generated by incubation of the substratewith Exo III. Lanes 3 and 4 each contain reaction products generated inreactions which contained 200 units of Exo III; the reaction in lane 3was run in a buffer containing 0 mM KCl, that in lane 4 was run in abuffer containing 50 mM KCl. Lanes 5 and 6 each contain reactionproducts generated in reactions which contained 1.25 units of Exo III;the reaction in lane 5 was run in a buffer containing 0 mM KCl, that inlane 6 was run in a buffer containing 50 mM KCl.

The results presented in FIG. 69 demonstrate that Exo III generated adistinct cleavage pattern when incubated with a single-stranded DNAsubstrate. The pattern generated by Exo III was entirely distinct fromthat generated by the CLEAVASE BN enzyme. The results shown in FIG. 69also show that significant differences in the cleavage pattern generatedby Exo III were observed depending on the concentrations of both theenzyme and KCl included in the reactions.

c) Ability of Alternative Enzymes to Identify Single Base Changes

In sections and a) and b) above it was shown that enzymes other thanCLEAVASE BN enzyme could generate a distinct pattern of cleavagefragments when incubated in the presence of a nucleic acid substrate.Because both Tth DNAP and E. coli Exo III generated distinct cleavagepatterns on single-stranded DNA, the ability of these enzymes to detectsingle base changes present in DNA substrates of the same size wasexamined. As in Example 11, the human tyrosinase gene was chosen as amodel system because numerous single point mutations have beenidentified in exon 4 of this gene.

Three single-stranded substrate DNAs were prepared; all three substratescontained a biotin label at their 5′ end. The wild type substratecomprises the 157 nucleotide fragment from the sense strand of the humantyrosinase gene (SEQ ID NO:47). Two mutation-containing substrates wereused. The 419 substrate (SEQ ID NO:54) and the 422 substrate (SEQ IDNO:55), both of which are described in Example 11. Single-stranded DNAcontaining a biotin label at the 5′ end was generated for each substrateusing asymmetric PCR as described in Example 10a with the exception thatthe single-stranded PCR products were recovered from the gel rather thanthe double-stranded products.

Cleavage reactions were performed as follows. Each substrate DNA(approximately 100 fmoles) was placed in a 200 μl thin wallmicrocentrifuge tubes (BioRad, Richmond, Calif.) in 5 μl of 1×CFLPbuffer with 1.33 mM MnCl₂ (to yield a final concentration of 1 mM). A noenzyme control was set up with the wild type DNA fragment in paralleland incubated at 65° C. for each of the indicated time points, with thedifferences that sterile distilled water was substituted for CLEAVASE BNenzyme and all reaction components were added prior to denaturation at95° C. The reaction tubes were brought to 95° C. for 5 seconds todenature the substrates and then the tubes were quickly cooled to 65° C.for the reactions containing Tth DNAP and 37° C. for the reactionscontaining Exo III.

Cleavage reactions were started immediately by the addition of a dilutedenzyme mixture containing 1.25 units of the enzyme either Tth DNAP orExo III in 5 μl of 1×CFLP buffer without MnCl₂. The enzyme solution wasbrought to room temperature before addition to the cleavage reaction.After 5 minutes at 65° C., the reactions were stopped by the addition of8 μl of stop buffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue,0.05% xylene cyanol). The samples were heated to 72° C. for 2 minutesand 7 μl of each reaction were resolved by electrophoresis through a 10%polyacrylamide gel (19:1 cross-link), with 7 M urea, in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated and overlaid with anylon membrane, as described in example 10a. The DNA was transferred tothe membrane and the membrane was dried, blocked in 1× I-BLOCK (Tropix,Bedford, Mass.), conjugated with streptavidin-alkaline phosphatase(United States Biochemical), washed, reacted with CDPSTAR reagent(Tropix, Bedford, Mass.), and exposed to X-ray film as described inExample 22a with the exception that the distilled water washes wereomitted. The results are presented in FIG. 70.

In FIG. 70, lanes 1-3 contain cleavage fragments generated by incubationof either the wild-type, mutant 419 and mutant 422 alleles of thetyrosinase gene, respectively, with Tth DNAP. Lanes 4-6 contain cleavagefragments generated by incubation of either the wild type, mutant 419and mutant 422 substrates, respectively, with Exo III in a buffercontaining 0 mM KCl. Lanes 7-9 contain cleavage fragments generated byincubation of either the wild type, mutant 419 and mutant 422substrates, respectively, incubated with Exo III in a buffer containing50 mM KCl. Lane 10 contains cleavage fragments generated by incubationof the wild type DNA substrate with CLEAVASE BN enzyme in a buffercontaining 0 mM KCl; this reaction provides a comparison of the patternsgenerated by the three different enzymes (i.e., CLEAVASE BN enzyme, TthDNAP and Exo III). Lane 11 contains the no enzyme control with the wildtype DNA substrate incubated in the presence of 50 mM KCl.

The results shown in FIG. 70 demonstrate that both Tth DNAP and Exo IIIwere able to detect single base changes in a single-stranded DNAsubstrate relative to a wild-type DNA substrate. The patterns generatedby Tth DNAP were comparable to those generated by CLEAVASE BN enzyme forall three DNA substrates (See FIG. 32 for a comparison of the patterngenerated by CLEAVASE BN enzyme).

The patterns generated by Exo III were entirely distinct from thosegenerated by enzymes derived from the genus Thermus (i.e., CLEAVASE BNenzyme and Tth DNAP). Furthermore, the pattern produced by cleavage ofthe DNA substrates by Exo III were distinct depending on whichconcentration of KCl was employed in the reaction (FIG. 70). A distinctpattern change was evident for the 419 mutant at both KClconcentrations. As shown in FIG. 70, at 0 mM KCl, a band appears in the40 nucleotide range in the 419 mutant (lane 5); at 50 mM KCl, the 419mutant contains an additional band in the 70 nucleotide range (lane 8).Pattern changes were not discernable for the 422 mutant (relative to thewild-type) in the Exo III digestions; this difference in the ability ofthe E. coli Exo III enzyme to detect single base changes could relate tothe relative positions of the changes with respect to secondarystructures that act as substrates for the structure specific cleavagereaction, and the position of the label (5′ or 3′ end) relative to thepreferred cleavage site (5′ or 3′), FIG. 71.

d) The Drosophila RrpI Enzyme can be Used to Generate Cleavage Patterns

Another protein in the Exo III family of DNA repair endonucleases, RrpIfrom Drosophila melanogaster (Nugent, M, Huang, S.-M., and Sander, M.Biochemistry, 1993: 32, pp. 11445-11452), was tested for its ability togenerate a distinct cleavage pattern on a single-stranded DNA template.Because its characteristics in the cleavage assay were unknown, thisenzyme was tested under a variety of buffer conditions. Varying amountsof this enzyme (1 ng or 30 ng) were incubated with approximately 100fmoles of the 157 nucleotide fragment 3 of the sense strand of exon 4 ofthe tyrosinase gene (SEQ ID NO: 47) in either 1 mM MnCl₂ or 5 mM MgCl₂and either 1×CFLP buffer, pH 8.2 or 1×CFLP buffer, pH 7.8, with 10 mMNaCl. Samples were heated to 95° C. and begun by the addition of adiluted enzyme mix containing either 1 or 30 ng of RrpI in 1×CFLPbuffer. Reactions were carried out at 30° C. for either 5 or 30 minutes.The results (data not shown) indicated that this enzyme generates aweak, but distinct cleavage pattern on a single-stranded DNA template.

e) The Rad1/Rad10 Complex can be Used to Generate Cleavage Patterns

The Rad1-Rad10 endonuclease (Rad1/10) from S. cerevisiae is a specific3′ endonuclease which participates in nucleotide excision repair inyeast. This enzyme is a heterodimer consisting of two proteins, Rad1 andRad10 Rad1 and Rad10 alone do not have enzymatic activity. Rad1/10recognizes structures comprising a bifurcated DNA duplex and cleaves thesingle-stranded 3′ arm at the end of the duplex (Bardwell, A. J et al.(1994) Science 265:2082). In this sense Rad1/10 shares the samesubstrate specificity as does the CLEAVASE BN enzyme. However, thecleavage products produced by Rad1/10 and CLEAVASE BN enzyme differ asthe Rad1/10 cleaves on the 3′ single-stranded arm of the duplex whileCLEAVASE BN enzyme cuts on the 5′ single-stranded arm.

FIG. 71 provides a schematic drawing depicting the site of cleavage bythese two enzymes on a bifurcated DNA duplex (formed by the hairpinstructure shown). In FIG. 71, the hairpin structure at the top shows thesite of cleavage by a 5′ nuclease (e.g., CLEAVASE BN enzyme). Thehairpin structure shown at the bottom of FIG. 71 shows the site ofcleavage by an enzyme which cleaves at the 3′ single-stranded arm (e.g.,Rad1/10). Enzymes which cleave on the 5′ single-stranded arm arereferred to as CLEAVASE 5′ enzymes; enzymes which cleave on the 3′single-stranded arm are referred to as CLEAVASE 3′ enzymes.

In order to determine whether the Rad1/10 protein is able to detectsingle base changes in DNA substrates, the cleavage patterns created bycleavage of DNA substrates by the Rad1/10 and CLEAVASE BN enzymes werecompared. In this comparison the following substrates were used. The 157nucleotide fragment from the wild type (SEQ ID NO:47), the 419 mutant(SEQ ID NO:54) and the 422 mutant (SEQ ID NO:55) alleles derived fromthe sense strand of exon 4 of the human tyrosinase gene was generatedcontaining a biotin label at the 5′ end as described in Example 11.

The Rad1 and Rad10 proteins were generously provided by Dr. Errol C.Friedberg (The University of Texas Southwestern Medical Center, Dallas).The Rad1/10 complex was prepared by mixing Rad1 and Rad10 proteins in 1×dilution buffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-HCl, pH 8.0, 50 mMKCl, 10 μg/ml BSA) to achieve a final concentration of 0.1 mM of eachprotein.

Cleavage reactions using the Rad1/10 endonuclease were performed asfollows. The substrate DNA and 15 ng (0.1 pmole) of Rad1/10 complex in 1μl of 1× dilution buffer were mixed on ice in 10 μl of 1×CFLP buffer pH7.8, 1 mM MnCl₂. The reaction was then incubated at 37° C. for 5minutes. The cleavage reaction was stopped by addition of 6 μl of stopbuffer.

Cleavage reactions using the CLEAVASE BN enzyme were done exactly asdescribed above for the Rad1/10 cleavages with the exception that 10 ngof the CLEAVASE BN enzyme was added and the incubation at 37° C. wasperformed for 3 minutes. Uncut or no enzyme controls were run for eachsubstrate DNA and were prepared as described for the reactionscontaining enzyme with the exception that sterile water was added inplace of the enzyme (data not shown).

The cleavage products (3 μl each) were separated by electrophoresisthrough a 10% denaturing polyacrylamide gel, transferred to a membraneand visualized as described in Example 21. The resulting autoradiographis shown in FIG. 72.

FIG. 72 shows the resulting patterns corresponding to the cleavageproducts of the sense strand of the wild type tyrosinase substrate (SEQID NO:47) (lanes 1 and 4), the 419 mutant (SEQ ID NO:54) (lanes 2 and 5)and the 422 mutant (SEQ ID NO:55) (lanes 3 and 6). Lanes 1-3 show thecleavage pattern created by incubation of the three substrate DNAs withthe CLEAVASE BN enzyme and lanes 4-6 show cleavage patterns created byincubation of the three substrate DNAs with the Rad1/10 enzyme. Lanesmarked “M” contain molecular weight markers prepared as described inExample 10.

The results shown in FIG. 72 demonstrate that the Rad1/10 enzyme wasable to produce distinctive cleavage patterns from the substrate DNAs(lanes 4-6); the average product length produced by cleavage of thesubstrate was longer than that produced by CLEAVASE BN enzyme.Importantly, the results shown in FIG. 72 demonstrate that the singlebase substitutions found in the mutant tyrosinase substrates resulted inthe production of specific changes in the otherwise similar cleavagepatterns of tyrosinase substrates (compare lanes 5 and 6 with lane 4).Note that in the digestion of the mutant 419 substrate with Rad1/10, thebands below about 40 nucleotides have lower intensity and one band isabsent, when compared to wild-type, while in the digest of the mutant422 substrate several new bands appear in the range of 42-80nucleotides. Since both enzymes were tested using the same reactionconditions, these results show that Rad1/10 was able to detect the samedifferences in DNA secondary structure that were recognized by CLEAVASEBN enzyme. Rad1/10 generates a different cleavage pattern relative tothat produced by CLEAVASE BN enzyme, since cleavage takes place at the3′ end of DNA hairpins producing inherently longer fragments when thesubstrate contains a 5′ end label.

Example 28 Detection of Mutations in the Human β-Globin Gene UsingDouble-Stranded DNA Substrates

The results shown in Example 15 demonstrated that single base changes infragments of the β-globin gene can be detected by cleavage ofsingle-stranded DNA substrates with the CLEAVASE BN enzyme. In thisexample it is shown that mutations in the β-globin gene can be detectedby cleavage of double-stranded DNA substrates using the CLEAVASE BNenzyme.

Double-stranded substrate DNA comprising 536 by fragments derived fromthe wild-type β-globin gene (SEQ ID NO:69), mutant 1 (SEQ ID NO:71) andmutant 2 (SEQ ID NO:72) were generated containing a 5′ biotin label onthe sense strand using the PCR. PCR amplification of these substrateswas done as described in Example 15a. Gel purification and isolation ofdouble-stranded fragments was performed as described in Example 21a.

The cleavage reactions were performed as described in Example 21c.Briefly, 2 μl of stock DNA (80 ng) in 10 mM Tris-HCl pH 8.0, 0.1 mM EDTAwas mixed with 3 μl H₂O and denatured at 95° C. for 20 seconds. Thedenatured DNA was cooled to 70° C. and a mixture consisting of 2 μl of5×CFLP buffer pH 7.5, 2 μl of 2 mM MnCl₂ and 1 μl (25 ng) of the enzymeCLEAVASE BN in dilution buffer was added to start the cleavage reaction.The cleavage reactions were stopped after 1 minute by the addition of 6μl of stop buffer. Control uncut reactions were performed as describedabove with the exception that of 1 μl of H₂O was used in place of 1 μlof the CLEAVASE BN enzyme. The cleavage products (5 μl each) wereseparated by electrophoresis through a 6% denaturing polyacrylamide gel,transferred to a membrane and visualized as described in Example 21. Theresulting autoradiograph is shown in FIG. 73.

FIG. 73 shows the cleavage patterns which correspond to the cleavage ofthe sense strand of the wild type β-globin 536 by fragment (lane 4),mutant 1 fragment (lane 5) and mutant 2 fragment (lane 6). Lanes 1-3show the uncut controls for wild-type, mutant 1 and mutant 2 substrates,respectively. The lane marked “M” contains biotinylated molecular weightmarkers prepared as described in Example 10.

As shown in FIG. 73, the base substitution present in mutant 1 resultsin a reduction in the intensity of a band which migrates close to theuncut DNA (lane 5), when compared to wild-type cleavage pattern. Thebase substitution present in mutant 2 results in the disappearance ofthe band present in the region just above major product band(approximately 174 nucleotides), when compared to the wild-type cleavagepattern.

For the double-stranded cleavage reactions described above, differentreaction conditions were used than those employed for the cleavage ofthe single-stranded β-globin DNA substrates described in Example 15. Theconditions employed for the cleavage of the double-stranded substratesused a lower MnCl₂ concentration, no KCl was added, a higher temperatureand shorter time course relative to the conditions used in Example 15.Although the cleavage patterns generated by cleavage of thedouble-stranded and single-stranded β-globin DNA were slightlydifferent, the positions of the pattern changes for mutants 1 and 2 aresimilar to those demonstrated in Example 15, and it was possible todetect the base substitutions in both double-stranded cases. Theseresults show that the subtle changes in DNA secondary structure causedby single base substitutions in larger DNA substrates can be detected bythe CLEAVASE BN enzyme whether a single- or double-stranded form of theDNA substrate is employed.

Example 29 Identification of Mutations in the Human β-Globin Gene CFLPPatterns of Unknowns by Comparison to an Existing Library of Patterns

The results shown in Examples 15 demonstrated that CLEAVASE BN enzymegenerates a unique pattern of cleavage products from each β-globinsubstrate tested. Differences in banding patterns were seen between thewild-type and each mutant; different banding patterns were seen for eachmutant allowing not only a discrimination of the mutants from thewild-type but also a discrimination of each mutant from the others. Todemonstrate that the products of the CLEAVASE enzyme reaction can becompared to previously characterized mutants for purposes ofidentification and classification, a second set of β-globin mutants werecharacterized and the CFLP patterns, by comparison to the set analyzedin Example 15, were used to determine if the mutants in the second setwere the same as any in the first set, or were unique to the second set.Although these isolates have all been described previously (specificreferences are cited for of these isolates at the end of this example),the experiment was performed “blind”, with the samples identified onlyby a number.

Five β-globin mutants were compared to the CFLP patterns from the firstset: the wild type β-globin gene (SEQ ID NO:69) or mutant 1 (SEQ IDNO:71), mutant 2 (SEQ ID NO:72) or mutant 3 (SEQ ID NO:70). Plasmids forcontaining these 5 new isolates were grown and purified, andsingle-stranded substrate DNA, 534 or 536 nucleotides in length, wasprepared for each of the 5 β-globin genes as described above in Example15a. Cleavage reactions were performed and reaction products wereresolved as described in Example 15; the resulting autoradiograph isshown in FIG. 74.

In FIG. 74, two panels are shown. Panel A shows the reaction productsfrom the β-globin isolates described in Example 15 (and as seen in FIG.43). Panel B shows the reaction products of the five additionalisolates, numbered 4, 5, 6, 7 and 8. The lanes marked “M” containbiotinylated molecular weight markers prepared as described in Example10.

By comparison to the CFLP patterns shown in Panel A, the isolates shownin Panel B can be characterized. It can be seen that the banding patternof isolate 4 (Panel B, lane 1) is the same as was seen for the wild-typeβ-globin substrate shown in Panel A (lane 1); isolate 8 (Panel B, lane5) is comparable to the previously characterized mutant 3 (Panel A, lane4); isolate number 6 (Panel B, lane 3) has changes in two areas of thepattern and appears to have features of both isolates 2 (Panel A, lane3) and 3 (Panel A, lane 4); isolates 5 and 7 (Panel B, lanes 2 and 4,respectively) appear to be identical, and they show a pattern not seenin panel A.

To confirm the relationships between the different isolates, theidentities of the mutations were then determined by primer extensionsequencing using the FMOL DNA Sequencing System (Promega Corp., Madison,Wis.) using the PCR primers (5′-biotinylated KM29 primer (SEQ ID NO:67)and 5′-biotinylated RS42 primer (SEQ ID NO:68)), according to themanufacturer's protocol. The sequencing reactions were visualized by thesame procedures used for the β-globin CFLP reactions, as described inExample 15b.

The two isolates that matched members of the original set by CFLPpattern analysis matched by sequence also. Isolate 4 is identical to thewild type sequence (SEQ ID NO: 69); isolate 8 is a duplicate of mutant 3(SEQ ID NO: 70).

Isolate 6 appears by CFLP pattern to have changes similar to both mutant2 and mutant 3 of the original set. The sequence of mutant 6 (SEQ IDNO:82) reveals that it shares a one base change with mutant 3, a silentC to T substitution in codon 3. Mutant 6 also has a G-to-A substitutionin codon 26, only 4 bases downstream of that found in mutant 2 (SEQ IDNO: 72). This mutation has been shown to enhance a cryptic splice sitecausing a fraction of the mRNA to encode a nonfunctional protein (Orkin,S. H., et al. (1982) Nature, 300:768). It is worthy of note that whilemutant 6 and mutant 2 both showed alteration in the band that migratesat about 200 nucleotides (e.g., the band is missing or weak in mutant 2but appears to be split into 3 weak bands in mutant 6) these changes arenot of identical appearance. These CFLP changes, caused by mutationsfour nucleotides apart, are distinguishable from each other.

The last two isolates, 5 and 7, had the same sequence (SEQ ID NO:83),and revealed a single base substitution within the first intron, at IVSposition 110. This mutation is associated with abnormal splicing leadingto premature termination of translation of the β-globin protein (R. A.Spritz et al. (1981) Proc. Natl. Acad. Sci. USA, 78:2455). It is worthyof note that the band that disappears in the CFLP patterns for thesemutants (at approximately 260 nucleotides, as compared to the sizemarkers) is between the indicative bands in the mutant 1 (atapproximately 400 nucleotides) and mutant 2 (at approximately 200nucleotides) CFLP patterns, and the actual mutation (at nucleotide 334from the labeled 5′ end) is between those of mutants 1 and 2, atnucleotides 380 and 207, respectively. Thus, the CFLP analysis not onlyindicated the presence of a change, but also gave positional informationas well.

From the results shown in FIG. 74, the unique pattern of cleavageproducts generated by CLEAVASE BN enzyme from each of the first four(wild type plus three variants) β-globin substrates tested was used asreference to characterize additional β-globin isolates. The bandingpatterns show an overall “familial” similarity, with subtle differences(e.g., missing or shifted bands) associated with each particularvariant. Differences in banding patterns were seen between the wild-typeand each mutant; different banding patterns were seen for each mutantallowing not only a discrimination of the mutant from the wild-type butalso a discrimination of each mutant from the others.

Example 30 Effect of the Order of Addition of the Reaction Components onthe Double-Stranded Cleavage Pattern

The cleavage reaction using a double-stranded DNA substrate can beconsidered a two-step process. The first step is the denaturation of theDNA substrate and the second step is the initiation of the cleavagereaction at the target temperature. As it is possible that the resultingcleavage pattern may differ depending on the conditions present duringdenaturation (e.g., whether the DNA is denatured in water or in abuffer) as well as on the conditions of reaction initiation (e.g.,whether the cleavage reaction is started by the addition of enzyme orMnCl₂) the following experiment was performed.

To study the effect of the addition of the reaction components on theresulting cleavage pattern, all possible mixing combinations for 4reaction components (i.e., DNA, CFLP buffer, MnCl₂ and the CLEAVASE BNenzyme) were varied. A single DNA substrate was used which comprised the536 by fragment derived from the wild-type β-globin gene (SEQ ID NO:69).The substrate DNA contained a biotin label at the 5′ end of the sensestrand and was prepared as described in Example 28.

The substrate was cut in 8 different cleavage reactions which employeddifferent combinations for the addition of the reaction components atthe denaturing and initiation steps. These reactions are describedbelow.

FIG. 75 shows the resulting patterns generated by cleavage of the sensestrand of the wild-type β-globin 536-bp substrate (SEQ ID NO:69). Inlane 1, the substrate DNA (40 fmoles of DNA in 1 μl of 10 mM Tris-HCl,pH 8.0, 0.1 mM EDTA mixed with 5 μl H₂O) was denatured at 95° C. for 10seconds, cooled to 55° C. and the reaction was started by the additionof a mixture containing 2 μl of 5×CFLP buffer with 150 mM KCl, 1 μl of 2mM MnCl₂ and 1 μl (50 ng) of the CLEAVASE BN enzyme. In lane 2, the DNAwas denatured in the presence of 2 μl of 5×CFLP buffer and reaction wasstarted at 55° C. by the addition of 1 μl MnCl₂ and 1 μl (50 ng) of theCLEAVASE BN enzyme. In lane 3, the DNA was denatured in the presence ofMnCl₂ and the reaction was started with addition of the buffer and theenzyme. In lane 4, the denaturation mixture included the substrate DNAand the enzyme and the reaction was started with addition of the bufferand MnCl₂. In lane 5, the substrate DNA was denatured in the presence ofCFLP buffer and MnCl₂ and then the enzyme was added at 55° C. In lane 6,the substrate DNA was denatured in the presence of CFLP buffer and theenzyme and then MnCl₂ was added at 55° C. Lane 7 shows the uncutcontrol. In lane 8, the DNA was denatured in the presence of the enzymeand MnCl₂ and then the buffer was added at 55° C. In lane 9, thesubstrate DNA was denatured in the presence of the enzyme, MnCl₂ and theCFLP buffer and then the mixture was incubated at 55° C. for 5 minutes.The lane marked “M” contains biotinylated molecular weight markersprepared as described in Example 10.

In all cases reaction was stopped by addition of 6 μl of stop buffer.The reaction products (5 μl each) were resolved by electrophoresisthrough a 10% denaturing polyacrylamide gel and the DNA was transferredto a membrane and visualized as described in Example 21. The resultingautoradiograph is shown in FIG. 75.

The results shown in FIG. 75 demonstrate that most ofdenaturation-initiation protocols employed generated identical cleavagepatterns with the exception of the reaction shown in lane 3. In thereaction shown in lane 3, the DNA was denatured in the presence of MnCl₂and in the absence of CFLP buffer. In the cases where the enzyme andMnCl₂ were added before the denaturation step (lanes 8,9) no labeledmaterial was detected. In these cases the label was released in a formof short DNA fragments which were produced as a result of nibbling(i.e., the exonucleolytic removal) of the label from the 5′ end of thedouble-stranded DNA template.

The results shown in FIG. 75 demonstrate that the order of addition ofthe reaction components has little effect upon the cleavage patternproduced with the exception that 1) the DNA should not be denatured inthe presence of MnCl₂ but in the absence of any buffering solution and2) the CLEAVASE BN enzyme and MnCl₂ should not be added together to theDNA prior to the denaturation step. Under these two exceptionalconditions, the 5′ label was removed from the 5′ end of the substrate bythe enzyme resulting in a loss of the signal.

Example 31 Detection of Mutations in Human p53 Gene by CLEAVASE FragmentLength Polymorphism (CFLP) Analysis

The results shown in preceeding examples demonstrated that the CFLPreaction could detect single base changes in fragments of varying sizefrom the human β-globin and tyrosinase genes and that the CFLP reactioncould be used to identify different strains of virus. The ability of theCLEAVASE enzyme reaction to detect single base changes in the humantumor suppressor gene p53 was next examined. Mutation of the human p53gene is the most common cancer-related genetic change; mutations in thep53 gene are found in about half of all cases of human cancer.

The ability of the CLEAVASE BN enzyme to cleave DNA fragments derivedfrom the human p53 gene and to detect single base changes in fragmentsof the same size was examined. Plamsids containing cDNA clonescontaining either wild type or mutant p53 sequences were used togenerate templates for analysis in the CFLP reaction. The p53 gene isquite large, spanning 20,000 base pairs and is divided into 11 exons.The use of a template derived from a cDNA allows for maximization of theamount of protein-encoding sequence that can be examined in a DNAfragment of a given size.

The nucleotide sequence of the coding region of the wild type human p53cDNA gene is listed in SEQ ID NO:92. The nucleotide sequence of thecoding region of the mutant 143 human p53 cDNA gene is listed in SEQ IDNO:93. The nucleotide sequence of the coding region of the mutant 249(silent) human p53 cDNA gene is listed in SEQ ID NO:94. A 601 nucleotidefragment spanning exons 5 through 8 was generated from each of thesethree p53 cDNAs as follows.

a) Preparation of the Substrate DNA

Six double stranded substrate DNAs were prepared for analysis in theCFLP reaction. The substrates contained a biotin label at either their5′ or 3′ end. The wild type substrate comprises a 601 nucleotidefragment spanning exons 5 through 8 of the cDNA sequence of the humanp53 gene (SEQ ID NO:92) (Baker, S. J. et al., Science (1990) 249:912).Two mutation containing substrates were used. The mutant 143 substrate(SEQ ID:93) is derived from a p53 mutant V143A which contains a valine(GTG) to alanine (GCG) substitution; this mutation differs from the wildtype p53 exon 5-8 fragment by a single nucleotide change (Baker, S. J.et al., Science (1990) 249:912). The mutant 249 (silent) substrate isderived from a p53 mutant which contains a single base change at aminoacid 249, from AGG to AGA (SEQ ID NO:94). This single base change doesnot result in a corresponding amino acid change and is thereforereferred to as a silent mutation.

The 601 by double stranded PCR fragments were generated as follows. Theprimer pair 5′-TCTGGGCTTCTTGCATTCTG (SEQ ID NO:95) and5′-GTTGGGCAGTGCTCGCTTAG (SEQ ID NO:96) were used to prime the PCRs. Thesynthetic primers were obtained from Integrated DNA Technologies(Coralville, Iowa). The primers were biotinylated on their 5′ ends withthe Oligonucleotide Biotinylation Kit purchased from USB-Amersham(Cleveland, Ohio) according to the manufacturers' protocols. When thesense strand was to be analysed in the CFLP reaction, the primer listedin SEQ ID NO:95 was labeled at the 5′ end with the biotin. When theanti-sense strand was to be analysed in the CFLP reaction, the primerlisted in SEQ ID NO:96 was labeled at the 5′ end with the biotin.

The target DNA used in the PCR for the generation of the 601 by fragmentderived from the wild type p53 cDNA was the plasmid CMV-p53-SN3 (Baker,S. J. et al., supra); this plasmid contains the coding region listed inSEQ ID NO:92. The target for the generation of the 601 by fragmentderived from the mutant 143 was the plasmid CMV-p53-SCX3 (Baker, S. J.et al., supra); this plasmid contains the coding region listed in SEQ IDNO:93. REF). The target for the generation of the 601 by fragmentderived from mutant 249 (silent) was the plasmid LTR 273 His (Chen, P.L. et al., Science (1990) 250:1576); this plasmid contains the codingregion listed in SEQ ID NO:94. DNA was prepared from bacteria harboringeach plasmid (plasmid DNA was isolated using standard techniques). The601 by PCR products were prepared as follows.

The symmetric PCR reactions contained 50 ng of plasmid DNA, 50 pmolesprimer 5′-TCTGGGCTTCTTGCATTCTG (SEQ ID:95), 50 pmoles of primer5′-GTTGGGCAGTGCTCGCTTAG (SEQ ID:96), 50 μM each dNTP, 20 mM Tris-Cl, pH8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05% NONIDET P-40(NP40) in a reaction volume of 95 μl. The reaction mixtures wereoverlaid with 50 μl CHILL OUT (MJ Research, Watertown, Mass.) and thetubes were heated to 95° C. for 2.5 min. Taq DNA polymerase (PromegaCorp., Madison, Wis.) was then added as 1.25 units of enzyme in 5 μl of20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and0.05% NONIDET P-40. The tubes were then heated to 95° C. for 45 seconds,cooled to 55° C. for 45 seconds and heated to 72° C. for 75 seconds for34 cycles with a 5 min incubation at 72° C. after the last cycle.

The PCR products were gel purified as follows. The products wereprecipitated by the addition of NaCl to a final concentration of 0.4M,20 μg glycogen carrier and 500 μl ethanol. The DNA was pelleted bycentrifugation and the PCR products were resuspended in 25 or 50 μlsterile distilled water to which was added an equal volume of a solutioncontaining 95% formamide, 20 mM EDTA and 0.05% each xylene cyanol andbromophenol blue. The tubes were then heated to 85° C. for 2 min and thereaction products were resolved by electrophoresis through a 6%polyacrylimide gel (19:1 cross-link) containing 7 M urea in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The DNA wasvisualized by ethidium bromide staining and the 601 by fragments wereexcised from the gel slices by passive diffusion overnight into asolution containing 0.5 M NH₄OAc, 0.1% SDS and 0.1% EDTA. The DNA wasthen precipitated with ethanol in the presence of 4 μg of glycogencarrier. The DNA was pelleted, resuspended in sterile distilled waterand reprecipitated by the addition of NaCl to a final aqueousconcentration of 0.2 M and 80% ethanol. After the second precipitation,the DNA was pelleted and resuspended in 30 μl sterile distilled water orTE (10 mM Tris-Cl, pH 8.0 and 0.1 mM EDTA).

The nucleotide sequnence of these 601 by templates are listed in SEQ IDNOS:97-102. The sense strand of the 601 nucleotide wild type fragment islisted in SEQ ID NO:97. The anti-sense strand of the 601 nucleotide wildtype fragment is listed in SEQ ID NO:98. The sense strand of the 601nucleotide mutant 143 fragment is listed in SEQ ID NO:99. The anti-sensestrand of the 601 nucleotide mutant 143 fragment is listed in SEQ IDNO:100. The sense strand of the 601 nucleotide mutant 249 (silent)fragment is listed in SEQ ID NO:101. The anti-sense strand of the 601nucleotide mutant 249 (silent) fragment is listed in SEQ ID NO:102.

b) Cleavage Reaction Conditions

Cleavage reactions comprised approximately 100 fmoles of the resultingdouble stranded substrate DNAs (the substrates contained a biotin moietyat the 5′ end of the sense or antisense strand) in a total volume of 5μl of sterile distilled water. The reactions were heated to 95° C. for15 seconds to denature the substrates and then quickly cooled to 50° C.(this step allows the DNA to assume its unique secondary structure byallowing the formation of intra-strand hydrogen bonds betweencomplimentary bases).

The reactions were performed in either a thermocycler (MJ Research,Watertown, Mass.) programmed to heat to 95° C. for 15 seconds and thencooled immediately to 50° C.

Once the tubes were cooled to the reaction temperature of 50° C., thefollowing components were added: 5 μl of a diluted enzyme mix containing0.2 μl of CLEAVASE BN enzyme (50 ng/μl 1× CLEAVASE enzyme DilutionBuffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-Cl, pH 8.0, 50 mM KCl, 10μg/ml BSA)); 1 μl of 10×CFLP reaction buffer (100 mM MOPS, pH 7.5, 0.5%NP 40, 0.5% TWEEN 20), and 1 μl of 2 mM MnCl₂.

A no enzyme control (10 μl) was set up in parallel for each PCR fragmentexamined; this control differed from the above reaction mixture only inthat sterile distilled water was substituted for CLEAVASE BN enzyme.Reactions were stopped after 3 minutes by the addition of 8 μl of stopbuffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue, 0.05% xylenecyanol).

The samples were then heated to 85° C. for 2 minutes and 4 μl of eachreaction mixture were resolved by electrophoresis through a 6%polyacrylimide gel (19:1 cross-link), with 7M urea, in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated allowing the gel toremain flat on one plate. A 0.2 μm-pore positively charged nylonmembrane (Schleicher and Schuell, Keene, N.H.), pre-wetted with 0.5×TBE(45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA), was laid on top of the exposedacrylamide gel. All air bubbles trapped between the gel and the membranewere removed. Two pieces of 3MM filter paper (Whatman) were then placedon top of the membrane, the other glass plate was replaced, and thesandwich was clamped with binder clips. Transfer was allowed to proceedovernight. After transfer, the membrane was carefully peeled from thegel and washed in 1× Sequencase Images Blocking Buffer (United StatesBiochemical) for two 15 minute intervals with gentle agitation. Threetenths of a ml of the buffer was used per cm² of membrane. Astreptavidin-alkaline phosphatase conjugate (SAAP, United StatesBiochemical, Cleveland, Ohio) was added to a 1:3000 dilution directly tothe blocking solution, and agitated for 15 minutes. The membrane waswashed 3 times (5 min/wash) in 1×SAAP buffer (100 mM Tris-HCl, pH 10; 50mM NaCl) with 0.1% SDS, using 0.5 mls/cm² of membrane. The membrane wasthen washed twice in 1×SAAP buffer without SDS, but containing 1 mMMgCl₂, drained thoroughly and placed in a heat sealable bag. Using asterile pipet tip, 0.05 ml/cm² of CDPSTAR reagent (Tropix, Bedford,Mass.) was added to the bag and distributed over the membrane for 5minutes. The bag was drained of all excess liquid and air bubbles. Themembrane was then exposed to X-ray film (Kodak XRP) for an initial 30minute exposure. Exposure times were adjusted as necessary forresolution and clarity. The result autoradiograph is shown in FIG. 79.

In FIG. 79, the lane marked “M” contains biotinlyated molecular weightmarkers. The marker fragments were purchased from Amersham (ArlingtonHeights, Ill.). Lanes 1-4 contain the reaction products from theincubation of double stranded DNA substrates in the absence of theCLEAVASE BN enzyme (i.e., uncut controls). Lane 1 contains the wild typefragment labeled on the sense strand of the 601 by PCR fragment. Lane 2contains the mutant 143 fragment labeled on the sense strand of the 601by PCR fragment. Lane 3 contains the wild type fragment labeled on theantisense strand of the PCR product. Lane 4 contains the fragmentencoding the silent mutation at amino acid 249 labeled on the antisensestrand of the PCR product. Lanes 5-8 contain the reaction products fromthe incubation of the 601 by double stranded substrates with CLEAVASE BNenzyme. Lane 5 contains products generated using the wild type fragmentlabeled on the sense strand; lane 6 contains products generated usingthe mutant 143 labeled on the sense strand. Lanes 7 and 8 containproducts generated using the wild type and mutant 249 (silent)substrates, respectively, labeled on the anti-sense strand.

The results shown in FIG. 79 demonstrate that a similar, but distinctlydifferent, pattern of cleavage products was generated by the digestionof wild type and mutant-containing templates by the CLEAVASE BN enzyme.Comparison of lanes 5 and 6 reveals a difference in the band pattern inthe 100 nucleotide range. Specifically, the strong band present in thewild type (at around 100 nucleotides) was missing in the V143A mutantwhile two bands immediately below this strong band were prominent in themutant and not evident in the wild type. In the 200 nucleotide range, apronounced doublet seen in the wild type is missing from the mutant,which instead contained a strong single band migrating slightly fasterthan the wild type doublet. Similarly, comparison of lanes 7 and 8revealed differences between the pattern generated from cleavage of theanti-sense strand of the wild type fragment and the mutant 249 (silent)fragment. In the 100 nucleotide range, the wild type fragment exhibiteda strong doublet whereas the upper band of this doublet was missing inthe mutant 249 (silent) fragment. In addition, two prominent bandspresent in the wild type pattern in the 150-180 by range were completelyabsent from the mutant 249 (silent) cleavage products.

Although each mutant fragment analyzed in FIG. 79 differs from the wildtype by only one of the 601 nucleotides, a unique pattern of cleavagefragments was generated for each. Furthermore, at least one patternchange occurred in each mutant in the immediate vicininty (i.e., within10-20 nucleotides) of the DNA sequence change. This experimentdemonstrates that CFLP analysis is capable of distinguishing thepresence of single base changes in PCR fragments containing exons 5through 8 of the p53 gene.

Example 32 Detection of Genetically Engineered Mutations in PCRFragments of the Human p53 Gene

The ability of the CLEAVASE BN enzyme to detect single base changesgenetically engineered into PCR fragments containing exons 5 through 8of the human p53 gene was analyzed. The single base changes introducedwere 1) a change from arginine (AGG) to serine (AGT) at amino acid 249(termed the R249S mutation) and 2) a change from arginine (CGT) tohistidine (CAT) at amino acid 273 (termed the R273H mutation). Both ofthese mutations have been found in human tumors and have been identifiedas mutational hot spots (Hollstein et al., Science 253:49 (1991)). TheR249S mutation is strongly correlated with exposure to aflatoxin Band/or infection with hepatitus B virus (Caron de Fromental and Soussi,Genes, Chromosomes and Cancer (1992) pp. 1-15). The R273H mutationarises as a result of a transition at a CpG dimucleotide. Suchtransitions account for approximately one-third of the known p53mutationns and are characteristic of a variety of tumor types (Caron deFromental and Soussi, supra; Hollstein et al., supra).

Plasmids containing the R249S and R273H mutations were engineeredaccording to a variation of a protocol described by R. Higuchi (in PCRTechnology: Principles and Applications for DNA Amplification, H. A.Ehrlich, Ed. (1989) Stockton Press, NY, pp. 61-70). This methodologyallows the generation of collection of plasmids containing DNA sequencescorresponding to known p53 mutations. The availability of thiscollection allows the generation of p53 “bar code” library whichcontains the CFLP patterns generated by cleavage of the p53 mutantsusing the CLEAVASE enzymes.

a) Construction of a 601 by PCR fragment Containing the R249S Mutation

To generate a 601 by fragment containing the R249S mutation, a 2-steprecombinant PCR was performed (see FIG. 78 for a schematicrepresentation of the 2-step recombinant PCR). In the first or“upstream” PCR, oligonucleotides 5′-TCTGGGCTTCTTGCATTCTG (SEQ ID NO:95)and 5′-GAGGATGGGAC TCCGGTTCATG (SEQ ID NO:103) were used to amplify a427 by fragment containing the G to T base change resulting in the R249Smutation; the sequence of the 427 by fragment is listed in SEQ IDNO:111. In the second or “downstream” PCR, oligonucleotide5′-CATGAACCGGAGTCCCATCCTCAC (SEQ ID NO:104) and 5′-GTTGGGCAGTGCTCGCTTAG(SEQ ID NO:96) were used to amplify a 196 by fragment containing thesame base change on the complementary strand; the sequence of the 196 byfragment is listed in SEQ ID NO:112.

For each PCR, 10 ng of a cDNA clone encoding the wild type p53 gene(coding region listed in SEQ ID NO:92) were used as the template in a 50μl PCR reaction. In the case of the upstream fragment, 10 ng of templatewere added to a tube containing 5 picomoles of the oligonucleotide5′-TCTGGGCTTCTTGCATT CTG (SEQ ID NO:95), 5 pmoles of the oligonucleotide5′-GAGGATGGGACTCC GGTTCATG (SEQ ID NO:103), 50 μM each dNTP, 20 mMTris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05%NONIDET P-40 (NP40) in a volume of 45 μl. For the downstream fragment,10 ng of the wild type template, plasmid CMV-p53-SN3 (Example 31) wereadded to 5 picomoles of the oligonucleotide 5′-CATGAACCGGAGTCCCATCCTCAC(SEQ ID NO:104) and 5 picomoles of the oligonucleotide5′-GTTGGGCAGTGCTCGCTTAG (SEQ ID NO:96), 50 μM each dNTP, 20 mM Tris-Cl,pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05% NONIDETP-40 (NP-40).

Tubes containing 45 μl of the above mixtures for each template to beamplified were overlaid with 50 μl CHILL OUT (MJ Research, Watertown,Mass.) and the tubes were heated to 95° C. for 2.5 min and then cooledto 70° C. Taq DNA polymerase (Promega) was then added as 1.25 units ofenzyme in 5 μl of 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, with0.05% TWEEN 20 and 0.05% NONIDET P-40. The tubes were then heated to 95°C. for 45 seconds, cooled to 55° C. for 45 seconds and heated to 72° C.for 75 seconds for 24 cycles with a 5 min incubation at 72° C. after thelast cycle.

The PCR products were gel purified as follows. Ten microliters of eachPCR product were mixed with 10 μl of stop buffer (95% formamide, 10 mMEDTA, 0.05% bromophenol blue, 0.05% xylene cyanol). The tubes were thenheated to 85° C. for 2 min and the reaction products were resolved byelectrophoresis through a 6% polyacrylimide gel (19:1 cross-link)containing 7 M urea in a buffer containing 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA (the polyacrylimide solutions used were freshly prepared).The DNA was visualized by ethidium bromide staining and the fragment wasexcised from the gel slice by passive diffusion overnight into asolution containing 0.5 M NH₄OAc, 0.1% SDS and 0.1% EDTA at 37° C.

Ten microliters of each eluted PCR product were combined to serve as therecombinant template to prime a second round of PCR. To this template,10 picomoles of 5′-biotin exon 8 primer (SEQ ID NO:96), 10 pmoles of5′-exon 5 primer (SEQ ID NO:95), 50 μM each dNTP, 20 mM Tris-Cl, pH 8.3,1.5 mM MgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05% NONIDET P-40(NP-40) were added. Tubes containing 90 μl of the above mixtures foreach template to be amplified were overlaid with 50 μl CHILL OUT (MJResearch, Watertown, Mass.) and the tubes were heated to 95° C. for 2.5min and then cooled to 70° C. Taq DNA polymerase (Promega) was thenadded as 2.5 units of enzyme in 5 μl of 20 mM Tris-Cl, pH 8.3, 1.5 mMMgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05% NONIDET P-40. The tubeswere then heated to 95° C. for 45 seconds, cooled to 47° C. to allow thetwo template molecules to anneal, then heated to 72° C. to allowextension of the primers by Taq DNA polymerase. Following this initialcycle of denaturation, annealing and extension, 25 cycles in which thereactions were heated to 95° C. for 45 seconds, cooled to 55° C. for 45seconds, and then heated to 72° C. for 1 minute were carried out,followed by a 5 min extension at 72° C. The fragments were then ethanolprecipitated and gel purified as described in Example 31.

b) Construction of a 601 by PCR Fragment Containing the R273H Mutation

To generate a 601 by fragment containing the R273H mutation, a 2-steprecombinant PCR was performed using the procedure described in sectiona) was used to simultaneously amplify PCR fragments encoding a singlebase change from arginine (CGT) to histidine (CAT) at amino acid 273. Inthe first or “upstream” PCR, oligonucleotide 5′-TCTGGGCTTCTTGCATTCTG-3′(SEQ ID NO:95) and 5′-GCACAAACATGCACCTCAAAGCT-3′ (SEQ ID NO:105) wereused to generate the 498 by fragment whose sequence is listed in SEQ IDNO:113. In the second or “downstream” PCR, oligonucleotide5′-CAGCTTTGAGGTGCATGTTTGT-3′ (SEQ ID NO:106) was paired witholigonucleotide 5′-GTTGGGCAGTGCTCGCTTAG-3′ (SEQ ID NO:96) to generate a127 nucleotide fragment whose sequence is listed in SEQ ID NO:114. TheDNA fragments were electrophoresed, eluted, combined and used to prime asecond round of PCR as described in section a) to generate a 601 by PCRproduct containing the R273H mutation.

c) Sequence Analysis of the 601 Nucleotide PCR Fragments

The recombinant 601-bp PCR products generated through this two step PCRprocedure were gel purified as described in Example 31. The PCR productswere sequenced using the FMOL DNA Sequencing System (Promega) inconjunction with oligonucleotide 5′-biotin-GTTGGGCAGTGCTCGCTTAG (SEQ IDNO:96) according to manufacturers' standard protocols to verify thepresence of the engineered mutations.

The nucleotide sequence corresponding to the sense strand of the 601nucleotide R249S mutant fragment is listed in SEQ ID NO:107. Theanti-sense strand of the 601 nucleotide R249S mutant fragment is listedin SEQ ID NO:108. The sense strand of the 601 nucleotide R273H mutantfragment is listed in SEQ ID NO:109. The anti-sense strand of the 601nucleotide R273H mutant fragment is listed in SEQ ID NO:110.

d) Cleavage Reactions

In order to generate ample quantities of DNA for subsequent CFLPanalysis, the 601 by fragments containing either the R249S or the R273Hmutation were used as templates in an additional round of PCR.Approximately 2 fmoles of each 601 by fragment were added to 20 pmolesof the primers corresponding to SEQ ID NOS:95 and 96 (SEQ ID NO:96contained a biotin on the 5′ end), 50 μM each dNTP, 20 mM Tris-HCl, pH8.3, 1.5 mM MgCl₂, 50 mM KCl, 0.05% TWEEN 20 and 0.05% NP40. Tubescontaining 90 μl of the above mixture were assembled for each templateto be amplified; the tubes were overlaid with 50 μl CHILL OUT (MJResearch, Watertown, Mass.) and the tubes were heated to 95° C. for 2.5min and then cooled to 70° C. Taq DNA polymerase (Promega) was thenadded as 2.5 units of enzyme in 5 μl of 20 mM Tris-Cl, pH 8.3, 1.5 mMMgCl₂, 50 mM KCl, with 0.05% TWEEN 20 and 0.05% NONIDET P-40. The tubeswere then heated to 95° C. for 45 seconds, cooled to 47° C. to allow thetwo template molecules to anneal, then heated to 72° C. to allowextension of the primers by Taq DNA polymerase. Following this initialcycle of denaturation, annealing and extension, 25 cycles in which thereactions were heated to 95° C. for 45 seconds, cooled to 55° C. for 45seconds, and then heated to 72° C. for 1 minute were carried out,followed by a 5 min extension at 72° C. The fragments were then ethanolprecipitated and gel purified as described in Example 31. The gelpurified fragments were then used in CFLP reactions as follows.

Cleavage reactions comprised approximately 100 fmoles of the resultingdouble stranded substrate DNAs (the substrates contained a biotin moietyat either the 5′ end of the sense or anti-sense strand) in a totalvolume of 5 μl (sterile distilled water was used to bring the volume to5 μl). The reactions were heated to 95° C. for 15 seconds to denaturethe substrates and then quickly cooled to 50° C. (this step allows theDNA to assume its unique secondary structure by allowing the formationof intra-strand hydrogen bonds between complimentary bases). Thereaction were performed in either a thermocycler (MJ Research,Watertown, Mass.) programmed to heat to 95° C. for 15 seconds and thencool immediately to 50° C. or the tubes were placed manually in a heatblock set at 95° C. and then transferred to a second heat block set at50° C.

Once the tubes were cooled to the reaction temperature of 50° C., 5 μlof a diluted enzyme mix containing 0.2 μl of CLEAVASE BN enzyme (50ng/μl 1× CLEAVASE Enzyme Dilution Buffer (0.5% NP40, 0.5% TWEEN 20, 20mM Tris-Cl, pH 8.0, 50 mM KCl, 10 μg/ml BSA)), 1 μl of 10×CFLP reactionbuffer (100 mM MOPS, pH 7.5, 0.5% NP 40, 0.5% TWEEN 20), and 1 μl of 2mM MnCl₂. A 10 μl no enzyme control was set up in parallel for each PCRfragment examined in which sterile distilled water was substituted forCLEAVASE BN enzyme. After 2 minutes at 50° C., the reactions werestopped by the addition of 8 μl of stop buffer (95% formamide, 10 mMEDTA, 0.05% bromophenol blue, 0.05% xylene cyanol).

The samples were heated to 85° C. for 2 minutes and 7 μl of eachreaction were resolved by electrophoresis through a 10% polyacrylimidegel (19:1 cross-link), with 7M urea, in a buffer containing 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated allowing the gel toremain flat on one plate. A 0.2 μm-pore positively charged nylonmembrane (Schleicher and Schuell, Keene, N.H.), pre-wetted with 0.5×TBE(45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA), was laid on top of the exposedacrylamide gel. All air bubbles trapped between the gel and the membranewere removed. Two pieces of 3MM filter paper (Whatman) were then placedon top of the membrane, the other glass plate was replaced, and thesandwich was clamped with binder clips. Transfer was allowed to proceedovernight. After transfer, the membrane was carefully peeled from thegel and washed in 1× Sequencase Images Blocking Buffer (United StatesBiochemical) for two 15 minute intervals with gentle agitation. Threetenths of a ml of the buffer was used per cm² of membrane. Astreptavidin-alkaline phosphatase conjugate (SAAP, United StatesBiochemical, Cleveland, Ohio) was added to a 1:3000 dilution directly tothe blocking solution, and agitated for 15 minutes. The membrane waswashed 3 times (5 min/wash) in 1×SAAP buffer (100 mM Tris-HCl, pH 10; 50mM NaCl) with 0.1% SDS, using 0.5 mls/cm² of membrane. The membrane wasthen washed twice in 1×SAAP buffer without SDS, but containing 1 mMMgCl₂, drained thoroughly and placed in a heat sealable bag. Using asterile pipet tip, 0.05 ml/cm² of CDPSTAR reagent (Tropix, Bedford,Mass.) was added to the bag and distributed over the membrane for 5minutes. The bag was drained of all excess liquid and air bubbles. Themembrane was then exposed to X-ray film (Kodak XRP) for an initial 30minute exposure. Exposure times were adjusted as necessary forresolution and clarity. The results are shown in FIG. 80.

In FIG. 80, the lane marked “M” contains biotinlyated molecular weightmarkers. The marker fragments were purchased from Amersham (ArlingtonHeights, Ill.) and include bands corresponding to lengths of 50, 100,200, 300, 400, 500, 700, and 1000 nucleotides. Lanes 1-4 contain thereaction products from the incubation of double stranded DNA substrateslabeled on the antisense strand in the absence of the CLEAVASE BNenzyme. Lane 1 contains the reaction products from the wild typefragment (SEQ ID NO:98); lane 2 contains the reaction products from theengineered R249S mutation (SEQ ID NO:108); lane 3 contains the reactionproducts from the 249 (silent) mutation (SEQ ID NO:102); lane 4 containsthe reaction products from the engineered R273H mutation (SEQ IDNO:110).

Lanes 5-8 contain the cleavage products generated from the sense strandeach of these templates when incubated in the presence of the CLEAVASEBN enzyme. Lane 5 contains the cleavage products from the wild typefragment (SEQ ID NO:97); lane 6 contains the cleavage products from theR249S fragment (SEQ ID NO:107); lane 7 contains the cleavage productsfrom the 249 (silent) mutant fragment (SEQ ID NO:101); lane 8 containsthe cleavage products from the R273S fragment (SEQ ID NO:109).

The results shown in FIG. 80 demonstrate that similar, but distinctlydifferent, patterns of cleavage were generated from each of thesetemplates containing single-base changes. Lane 6 shows the attenuationof bands in the 150-180 nucleotide range, as well as, the loss of a bandin the 100 nucleotide range when compared to the wild-type pattern shownin lane 5. In addition, lane 6 shows a new band appearing in the 140nucleotide range, and increased intensity in the top band of a doubletat about 120 nucleotides. Examination of the silent 249 mutant (lane 7)which differs from wild-type at the same nucleotide position as R249S(lane 6), revealed pattern differences relative to both the wild type(lane 5) as well as to the R249S (lane 6) mutation. Specifically,comparision to lane 5 shows an attenuation of bands in the 150-180nucleotide range as well as the loss of a band in the 100 nucleotiderange, as was seen in lane 6. However, the sample in lane 7 does notexhibit the additional band in the 140 nucleotide range, nor theincreased intensity in the top band of the doublet in the 120 nucleotiderange seen in lane 6. This result demonstrates that the CFLP techniqueis capable of distinguishing between changes to a different base at thesame nucleotide position.

Examination of the reaction products in lane 8 reveals the loss of aband in the 100 nucleotide range in the R273S fragment when compared tothe wild-type pattern in lane 5. This CFLP pattern is distinct fromthose in lanes 6 and 7, however, in that it does not show attenuation ofbands in the 150-180 nucleotide range; in this region of the gel thispattern is essentially indistinguishable from that generated from thewild type fragment.

The above results demonstrate that CFLP analysis can be used to detectclinically significant mutations in the human p53. Further, theseresults indicate that the CFLP technique is sufficiently sensitive todistinguish different base changes at the same position from oneanother, as well as from wild type. In addition these results show thatthe 2-PCR technique can be used to generate a collection of PCRfragments containing known p53 mutations; such a collection allows thegeneration of a p53 bar code library containing the CFLP patternsgenerated by different p53 mutations.

Example 33 Detection of the Presence of Wild Type and Mutant Sequencesin Mixed Samples

The ability of the CFLP reaction to detect the presence of differentalleles of the same sized PCR fragments in a mixed sample, such as mightbe found in heterozygous or otherwise heterogenous tissue, samples wasexamined.

PCR products containing a bitoin label on the sense strand were producedand purified as described in Example 31 for the wild type p53 (SEQ IDNO:97) and mutant 143 (SEQ ID NO:99) 601-bp fragments. Aliquots of thesesamples were diluted to a final concentration of approximately 12.5fmols/μl and mixed in different proportions to give a spectrum of ratiosof wild type to mutant DNA. Four microliters of the diluted DNA samples,for an approximate total of 50 fmols of DNA in each sample, mixed invarious combinations, were placed in microfuge tubes and heated to 95°C. for 15 seconds. The tubes were rapidly cooled to 50° C. and 6 μl of adiluted enzyme mix containing 0.2 μl of CLEAVASE BN enzyme (50 ng/μl 1×CLEAVASE Enzyme

Dilution Buffer (0.5% NP40, 0.5% TWEEN 20, 20 mM Tris-Cl, pH 8.0, 50 mMKCl, 10 μg/ml BSA)), 1 μl of 10×CFLP reaction buffer (100 mM MOPS, pH7.5, 0.5% NP 40, 0.5% TWEEN 20), and 1 μl of 2 mM MnCl₂. A 10 μl noenzyme control was set up in parallel for each PCR fragment examined,with the difference that sterile distilled water was substituted for theCLEAVASE BN enzyme. After 1.5 minutes at 50° C., the reactions werestopped by the addition of 8 μl of stop buffer (95% formamide, 10 mMEDTA, 0.05% bromophenol blue, 0.05% xylene cyanol). In addition, 4 μl ofwild type only as well as 4 μl of V143A only were analyzed by the samemethod for comparison to the mixed samples.

Samples were heated to 85° C. for 2 minutes and 7 μl of each reactionwere resolved by electrophoresis through a 10% polyacrylimide gel (19:1cross-link), with 7M urea, in a buffer containing 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated allowing the gel toremain flat on one plate. A 0.2 μm-pore positively charged nylonmembrane (Schleicher and Schuell, Keene, N.H.), pre-wetted with 0.5×TBE(45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA), was laid on top of the exposedacrylamide gel. All air bubbles trapped between the gel and the membranewere removed. Two pieces of 3MM filter paper (Whatman) were then placedon top of the membrane, the other glass plate was replaced, and thesandwich was clamped with binder clips. Transfer was allowed to proceedovernight.

After transfer, the membrane was carefully peeled from the gel andwashed in 1× Sequencase Images Blocking Buffer (United StatesBiochemical) for two 15 minute intervals with gentle agitation. Threetenths of a ml of the buffer was used per cm² of membrane. Astreptavidin-alkaline phosphatase conjugate (SAAP, United StatesBiochemical, Cleveland, Ohio) was added to a 1:3000 dilution directly tothe blocking solution, and agitated for 15 minutes. The membrane waswashed 3 times (5 min/wash) in 1×SAAP buffer (100 mM Tris-HCl, pH 10; 50mM NaCl) with 0.1% SDS, using 0.5 mls/cm² of membrane. The membrane wasthen washed twice in 1×SAAP buffer without SDS, but containing 1 mMMgCl₂, drained thoroughly and placed in a heat sealable bag. Using asterile pipet tip, 0.05 ml/cm² of CDPSTAR reagent (Tropix, Bedford,Mass.) was added to the bag and distributed over the membrane for 5minutes. The bag was drained of all excess liquid and air bubbles. Themembrane was then exposed to X-ray film (Kodak XRP) for an initial 30minute exposure. Exposure times were adjusted as necessary forresolution and clarity. The resulting autoradiograph is shown in FIG.81.

In FIG. 81, the lane marked “M” contains biotinylated molecular weightmarkers obtained from Amersham (Arlington Heights, Ill.) and includebands corresponding to lengths of 50, 100, 200, 300, 400, 500, 700, and1000 nucleotides. Lanes 1 and 2 contain the reaction products from theno enzyme controls for the wild type and V143A mutant fragments,respectively. Lane 3 contains cleavage products from the samplecontaining the wild type fragment only. Lane 4 contains cleavageproducts from wild type and mutant fragments mixed in a 1:1 ratio. Lane5 contains cleavage products from a reaction containing a 1:2 ratio ofwild type to mutant fragment. Lane 6 contains reaction products presentin a ratio of wild type to mutant of 1:9. Lane 7 contains cleavageproducts from a sample containing V143A mutant DNA only. Lane 8 containscleavage products mixed at a ratio of wild type to mutant of 2:1. Lane 9contains cleavage products mixed at a ratio of wild type to mutant 4:1.Lane 10 contains cleavage products mixed a ratio of wild type to mutantto 9:1.

The results shown in FIG. 81 demonstrate that the presence of differentalleles can be detected in a mixed sample. Comparison of lanes 4-6 andlanes 8-10 with either lane 3 or lane 7 demonstrates that the lanescontaining mixed reactions exhibit distinct differences from eithersample alone. Specifically, in the 100 nucleotide region, there is adoublet in the wild type sample that shifts in the mutant (seediscussion of FIG. 80 in Example 31). All three of these bands arepresent in the mixed samples (lanes 4-6 and lanes 8-10) whereas only oneor the other pair is detectable in lanes 3 and 7.

Example 34 Detection and Identification of Hepatitis C Virus Genotypesby CLEAVASE Fragment Length Polymorphism Analysis

Hepatitis C virus (HCV) infection is the predominant cause ofpost-transfusion non-A, non-B (NANB) hepatitis around the world. Inaddition, HCV is the major etiologic agent of hepatocellular carcinoma(HCC) and chronic liver disease world wide. Molecular biologicalanalysis of the small (9.4 kb) RNA genome has showed that some regionsof the genome are very highly conserved between isolates, while otherregions are subject to fairly rapid mutation. These analyses haveallowed these viruses to be divided into six basic genotype groups, andthen further classified into several sub-types (Altamirano et al., J.Infect. Dis. 171:1034 (1995)). These viral groups are associated withdifferent geographical areas, and accurate identification of the agentin outbreaks is important in monitoring the disease. While only genotype1 HCV has been observed in the United States, multiple HCV genotypeshave been observed in both Europe and Japan. HCV genotype has also beenassociated with differential efficacy of treatment with interferon, withGroup 1 infected individuals showing little response. The ability toidentify the genotype of HCV present in an infected individual allowscomparisons of the clinical outcomes from infection by the differenttypes of HCV, and from infection by multiple types in a singleindividual. Pre-screening of infected individuals for the viral typewill allow the clinician to make a more accurate diagnosis, and to avoidcostly but fruitless drug treatment.

In order to develop a rapid and accurate method of typing HCV present ininfected individuals, the ability of the CLEAVASE enzyme reaction todetect and distinguish between the major genotypes and subtypes of HCVwas examined. Plasmids containing DNA derived from the conserved 5′untranslated region of six different HCV RNA isolates were used togenerate templates for analysis in the CFLP reaction. The HCV sequencescontained within these six plasmids represent genotypes 1 (foursub-types represented; 1a, 1b, 1c and A 1c), 2 and 3. The nomenclatureof the HCV genotypes used is that of Simmonds et al. (as described inAltamirano et al., supra).

a) Generation of Plasmids Containing HCV Sequences

Six DNA fragments derived from HCV were generated by RT-PCR using RNAextracted from serum samples of blood donors; these PCR fragments were agift of Dr. M. Altamirano (University of British Columbia, Vancouver).These PCR fragments represent HCV sequences derived from HCV genotypes1a, 1b, 1c, A 1c, 2c and 3a.

The RNA extraction, reverse transcription and PCR were performed usingstandard techniques (Altamirano et al., J. Infect. Dis. 171:1034(1995)). Briefly, RNA was extracted from 100 μl of serum using guanidineisothiocyanate, sodium lauryl sarkosate and phenol-chloroform (Inchauspeet al., Hepatology 14:595 (1991)). Reverse transcription was performedaccording to the manufacturer's instructions using a GeneAmp rTh reversetranscriptase RNA PCR kit (Perkin-Elmer) in the presence of an externalantisense primer, HCV342. The sequence of the HCV342 primer is5′-GGTTTTTCTTTGAGGTTTAG-3′ (SEQ ID NO:115). Following termination of theRT reaction, the sense primer HCV7 (5′-GCGACACTCCACCATAGAT-3′ (SEQ IDNO:116)) and magnesium were added and a first PCR was performed.Aliqouts of the first PCR products were used in the second (nested) PCRin the presence of primers HCV46 (5′-CTGTCTTCACGCAGAAAGC-3′ (SEQ IDNO:117)) and HCV308 (5′-GCACGGTCTACGAGACCTC-3′ (SEQ ID NO:118)). ThePCRs produced a 281 by product which corresponds to a conserved 5′noncoding region (NCR) region of HCV between positions −284 and −4 ofthe HCV genome (Altramirano et al., J. Infect. Dis. 171:1034 (1995)).

The six 281 by PCR fragments were used directly for cloning or they weresubjected to an additional amplification step using a 50 μl PCRcomprising approximately 100 fmoles of DNA, the HCV46 and HCV308 primersat 0.1 μM, 100 μM of all four dNTPs and 2.5 units of Taq polymerase in abuffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂ and0.1% TWEEN 20. The PCRs were cycled 25 times at 96° C. for 45 sec., 55°C. for 45 sec. and 72° C. for 1 min. Two microliters of either theoriginal DNA samples or the reamplified PCR products were used forcloning in the linear pT7Blue T-vector (Novagen, Madison, Wis.)according to manufacturer protocol. After the PCR products were ligatedto the pT7Blue T-vector, the ligation reaction mixture was used totransform competent JM109 cells (Promega). Clones containing the pT7BlueT-vector with an insert were selected by the presence of colonies havinga white color on LB plates containing 40 μg/ml X-Gal, 40 μg/ml IPTG and50 μg/ml ampicillin. Four colonies for each PCR sample were picked andgrown overnight in 2 ml LB media containing 50 μg/ml carbenicillin.Plasmid DNA was isolated using the following alkaline miniprep protocol.Cells from 1.5 ml of the overnight culture were collected bycentrifugation for 2 min. in a microcentrifuge (14K rpm), thesupernatant was discarded and the cell pellet was resuspended in 50 μlTE buffer with 10 μg/ml RNAse A (Pharmacia). One hundred microliters ofa solution containing 0.2N NaOH, 1% SDS was added and the cells werelysed for 2 min. The lysate was gently mixed with 100 μl of 1.32 Mpotassium acetate, pH 4.8, and the mixture was centifugated for 4 min.in a microcentrifuge (14K rpm); the pellet comprising cll debris wasdiscarded. Plasmid DNA was precipitated from the supernatant with 200 μlethanol and pelleted by centrifugation a microcentrifuge (14K rpm). TheDNA pellet was air dried for 15 min. and was then redissolved in 50 μlTE buffer (10 mM Tris-HCl, pH 7.8, 1 mM EDTA).

To analyze the cloned HCV inserts, 1 μl of plasmid DNA (approximately 10to 100 ng) reamplified in a 50 μl PCR using the HCV46 and HCV308 primersas described above with the exception that 30 cycles of amplificationwere employed. The PCR products were separated by electrophoresis on a6% non-denaturing acrylamide gel (29:1 cross linked) in 0.5×TBE buffer;clones that gave rise to a 281 by PCR product were selected for furtheranalysis.

For sequencing purposes, plasmid DNA from selected clones was PEGpurified as follows. To 50 μl of plasmid DNA in TE buffer (approximately10-100 ng/μl), 25 μl of 5M NaCl and 10 μl 20% PEG (M.W.8,000; Fisher)was added, mixed well, and the mixture was incubated on ice for 1 hour.The mixture was then centrifuged for 5 min in a table-topmicrocentrifuge (at 14K rpm), the pellet was removed and an additional15 μl of 20% PEG was added to the supernatant. After incubation for 1hour on ice, a second pellet was collected by centrifugation, thesupernatant was discarded, and the pellet was redissolved in 20 μl H₂O.Two microliters of PEG-purified plasmid DNA (approximately 100 ng) wasused in cycle-sequencing reactions using the FMOL DNA Sequencing System(Promega, Madison, Wis.) according to manufacturer protocol, inconjunction with the HCV46 or HCV308 primers. The HCV46 or HCV308primers were biotinylated at the 5′ end using Oligonucleotide BiotinLabeling kit (Amersham, Arlington Heights, Ill.) prior to use in thesequencing reactions. Sequencing reactions were separated on 10%denaturing acrylamide gel, transferred on nylon membrane and visualizedas described in Example 21.

Alternatively, DNA sequencing was done using either the Blue-T1(5′-GATCTACTAGTCATATGGAT-3′ (SEQ ID NO:119)) and Blue-T2(5′-TCGGTACCCGGGGATCCGAT-3′ (SEQ ID NO:120)) primers labeled at the 5′end with tetra chloro fluorescein (TET) dye (Integrated DNATechnologies). In this case, the sequencing reactions were separated ona 10% denaturing acrylamide gel and the products were visualized using aFMBIO-100 Image Analyzer (Hitachi). The six HCV clones were termedHCV1.1, HCV2.1, HCV3.1, HCV4.2, HCV6.1 and HCV7.1; the double-strandedDNA sequence of these clones are listed in SEQ ID NOS:121-126,respectively. The sequence of the sense strand for each of the six HCVclones is shown as the top line in SEQ ID NOS:121-126. The sequence ofthe anti-sense strand for HCV clones HCV1.1, HCV2.1, HCV3.1, HCV4.2,HCV6.1 and HCV7.1 is listed in SEQ ID NOS:127-132, respectively.

The DNA sequences of each of the six HCV clones are aligned in FIG. 82.In FIG. 82, nucleotides which represent variations between the six HCVclones are indicated by bold type and underlining; dashes are used toindicate gaps introduced to maximize alignment between the sequences(necessary due to the insertion found in clone HCV4.2). This alignmentshows that these six HCV clones represent six different HCV genotypes.HCV1.1 represents a genotype 1c HCV; HCV2.1 represents a genotype 1aHCV; HCV3.1 represents a genotype 1b HCV; HCV4.2 represents a genotype1c HCV; HCV6.1 represents a genotype 2c HCV and HCV7.1 represents agenotype 3a HCV. For one sample, HCV4.2, an insertion of an “G”nucleotide was found at position 146 (relative to the protypical HCV;Altamirano et al., supra), since no insertion or deletions in the HCVNCR have been previously reported, a second independent clone derivedfrom the PCR products corresponding to HCV4 was sequenced. This secondHCV4 clone was found to have the same sequence as that shown for HCV4.2in FIG. 82.

b) Preparation of HCV Substrates

Six double stranded substrate DNA were prepared for analysis in the CFLPreaction. The substrates were labelled at the 5′ end of either the senseor the anti-sense strand by the use of labeled primers in the PCR topermit CFLP analysis of each strand of the HCV DNA substrates.

To prepare PCR products for CFLP analysis, the HCV46 and HCV308 primerswere 5′ end labeled with TMR dye using the ONLY BODIPY TMROligonucleotide Phosphate Labeling Kit (Molecular Probes, Inc., Eugene,Oreg.) according to manufacturer protocol. All six HCV 281 by NCRsequences were PCR amplified using 10 ng of template and 30 cycles ofamplification as described above in section a).

For sense strand analysis, the PCR was conducted using the HCV46 primer(SEQ ID NO:117) labeled with TMR and unlabeled HCV308 primer (SEQ IDNO:118). For antisense analysis, the PCR was conducted using unlabeledHCV46 primer (SEQ ID NO:117) and HCV308 primer (SEQ ID NO:118) labeledwith TMR. The PCR products were purified by electrophoresis on a 6%denaturing acrylamide gel and eluted overnight as described above inExample 21. The gel-purified DNA substrates were redissolved in 20 μlH₂O at an approximate concentration of 100 fmoles/μl.

c) Cleavage Reaction Conditions

Cleavage reactions comprised 1 μl of TMR-labeled PCR products(approximately 100 fmoles of the double-stranded substrates) in a totalvolume of 10 μl containing 1×CFLP buffer (10 mM MOPS, pH 7.5; 0.5% eachTWEEN 20 and NP-40) and 10 ng CLEAVASE BN enzyme. All components exceptthe MnCl₂ were assembled in a volume of 8 μl. The reactions were heatedto 95° C. for 15 seconds to denature the substrates and then quicklycooled to 55° C. The reaction were performed in either a thermocycler(MJ Research, Watertown, Mass.) programmed to heat to 95° C. for 15seconds and then cool immediately to 55° C. or the tubes were placedmanually in a heat block set at 95° C. and then transferred to a secondheat block set at 55° C.

Once the tubes were cooled to the reaction temperature of 55° C., thecleavage reaction was started by the addition of 2 μl of 1 mM MnCl₂.After 2 minutes at 55° C., the reactions were stopped by the addition of5 μl of a solution containing 95% formamide, 10 mM EDTA and 0.02% methylviolet.

Five microliters of each reaction mixture were heated at 85° C. for 2min, and where than resolved by electrophoresis through a 12% denaturingpolyacrylamide gel (19:1 cross link) with 7M urea in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. The gels were run at 33 watts for 1.5hours. The labeled reaction products were visualized using the FMBIO-100Image Analyzer (Hitachi), with the resulting fluoroimager scan shown inFIG. 83.

In FIG. 83, the CFLP patterns produced by cleavage of the six HCVsamples labeled on the sense strand are shown in lanes 1-6; the CFLPpatterns produced by cleavage of the six HCV samples labeled on theanti-sense strand are shown in lanes 7-12. The position of molecularweight markers is indicated on the left-hand side of the fluoroimagerscan by the large arrowheads; the size of the markers is indicated innucleotides.

The experiment presented in FIG. 83 demonstrates the ability of CFLPanalysis to differentiate six distinct hepatitis C viral subtypes. Thesix samples in the left hand side of the panel (lanes 1-6) were labeledon the 5′ end of the sense strand; the six on the right (lanes 7-12), onthe 5′ end of the antisense strand. The first four samples in each setall contain samples amplified from HCV type 1. Subtypes a, b, and c arerepresented, as is a single base deletion of type 1c (i.e., Δ1c).Analysis of either strand points out numerous similarities as well asseveral distinctive differences between the subtypes. Most notable amongthe similarities on the sense strand are prominent bands marked A, B andC. Specifically, whereas bands B and C are evident in the patternsgenerated from both subtypes 1a and 1b (and are, in fact, more prominentin subtype 1b than in 1a), they are barely visible in subtype 1c. BandA, though present in all 4 of these samples, is more prominent in thepatterns generated from subtypes 1c and 1a. Differences between subtypes2c and 3a vs. all of the subtype 1 samples, are evident in the regionbetween 50 and 100 nt (compare bands D and E) on the sense strand andbetween the 80 and 150 nt on the antisense strand (compare bands F-J).Viral type 2 gives rise to the most significantly altered CFLP pattern,while type 3 appears to be similar to type 1; these relationships appearto be consistent with the relative number of sequence differencesbetween the different isolates.

The results shown in FIG. 83 demonstrate that the CFLP method provides asimple and rapid method to determine the genotype of HCV strains. Thismethod will facilitate the diagnosis of HCV infection, permitappropriate treatment of HCV-infected patients, and aid in themoinitoring of HCV outbreaks.

Example 35 Detection of Mutations Associated With Antiobiotic Resistancein Mycobacterium tuberculosis

In the past decade there has been a tremendous resurgence in theincidence of tuberculosis in this country and throughout the world.Worldwide, the number of new cases reported annually is forecast toincrease from 7.5 million in 1990 to 10.2 million by the year 2000. Analarming feature of this resurgence in tuberculosis is the increasingnumbers of patients presenting with strains of M. tuberculosis which areresistant to one or more antituberculosis drugs (i.e., multi-drugresistant tuberculosis (MDR-TB)).

Resistance to either or both of the antibiotics rifampin (rif) andisoniazid (inh) is the standard by which M. tuberculosis strains arejudged to be multi-drug resistant. Both because of their potentbactericidal activities and because acquisition of primary resistance tothese drugs is rare (the spontaneous mutation rate of resistance torifampin is approximately 10⁻⁸ and to isoniazid, 10⁻⁸ to 10⁻⁹), untilvery recently, these two antibiotics were among the most powerfulfront-line drugs used to combat the advance and spread of tuberculosis.However surveys of tuberculosis patients in the U.S. reveal that as manyas one-third were infected with strains resistant to one or moreantituberculosis drugs; greater than 25% of the M. tuberculosis culturesisolated were resistant to isoniazid and 19% were resistant to bothisoniazid and rifampin (Frieden et al., New Eng. J. Med. 328:521(1993)).

As discussed above (Description of the Invention), resistance torifampin is associated with mutation of the rpoB gene in M.tuberculosis. While the exact mechanism of resistance to isoniazid isnot clear, the majority (as many as 80%) of inh^(r) mutations occur inthe katG and inhA genes of M. tuberculosis. To investigate whether CFLPanalysis could be used to detect mutations in the genes involved inMDR-TB, DNA fragments were amplified from the rpoB and katG genes of M.tuberculosis. DNA fragments derived from wild-type (i.e.,antibiotic-sensitive) or mutant (i.e., antibiotic-resistant) strains ofM. tuberculosis were subjected to CFLP analysis.

a) CFLP Analysis of Mutations in the RpoB Gene of M. tuberculosis

i) Generation of Plasmids Containing RpoB Gene Sequences

Genomic DNA isolated from wild-type M. tuberculosis or M. tuberculosisstrains containing mutations in the rpoB gene associated with rifampinresistance were obtained from Dr. T. Schinnick (Centers for DiseaseControl and Prevention, Atlanta, Ga.). The rifampin resistant strain #13(91-3083) contains a tyrosine residue at codon 451 of the rpoB gene inplace of the histidine residue found in the wild-type strain (i.e.,H451Y); this mutation is present in 28% of rifampin resistant TBisolates. The H451Y mutation is hereinafter referred to as mutant 1. Therifampin resistant strain #56 (91-2763) contains a luecine residue atcodon 456 of the rpoB gene in place of the serine residue found in thewild-type strain (i.e., S456L); this mutation is present in 52% ofrifampin resistant TB isolates. The S456L mutation is hereinafterreferred to as mutant 2.

A 620 by region of the TB rpoB gene was amplified using the PCR from DNAderived from the wild-type and mutant 1 and mutant 2 strains. Theprimers used to amplify the rpoB gene sequences were PolB-5A(5′-ATCAACATCCGGCCGGTGGT-3′ (SEQ ID NO:133)) and PolB-5B(5′-GGGGCCTCGCTACGGACCAG-3′ (SEQ ID NO:134)); these PCR primers amplifya 620 by region of the rpoB gene which spans both the H451Y and S456Lmutations (Miller et al., Antimicrob. Agents Chemother., 38:805 (1994)).The PCRs were conducted in a final reaction volume of 50 μl containingthe PolB-5A and PolB-5B primers at 1 μM, 1.5 mM MgCl₂, 20 mM Tris-HCl,pH 8.3, 50 mM KCl, 0.05% each TWEEN-20 and NONIDET P-40 and 60 μM of allfour dNTPs. The reaction mixture was heated at 95° C. for 3 min.Amplification was started by the addition of 2.5 units of Taq polymeraseand was continued for 35 cycles at 95° C. for 1 min, 60° C. for 1 minand 72° C. for 2 min.

To clone the PCR-amplified fragments, 1 μl of each PCR product was usedfor ligation in the linear pT7Blue T-vector (Novagen, Madison, Wis.).The ligation products were used to transform competent JM109 cells andclones containing pT7Blue T-vector with an insert were selected by whitecolor on LB plates containing 40 μg/ml X-Gal, 40 μg/ml IPTG and 50 μg/mlampicillin. For each PCR sample (i.e., wild-type and mutants 1 and 2),five independent colonies were picked and grown overnight in 2 ml of LBmedia containing 50 μg/ml carbenicillin. Plasmid DNA was isolated usingthe alkaline miniprep protocol described above in Example 34.

To analyze the cloned fragments, 1 μl of plasmid DNA from each clone wasamplified by PCR using 50 μl reaction containing the PolB-5A and PolB-5Bprimers at 1 μM, 1.5 mM MgCl₂, 20 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.05%each TWEEN-20 and NONIDET P-40, 60 μM of all 4 dNTPs and 2.5 units ofTaq polymerase. The PCRs were cycled 35 times at 95° C. for 1 min, 60°C. for 1 min and 72° C. for 2 min. The PCR products were separated byelectrophoresis on a 6% native polyacrylamide gel in 0.5×TBE buffer andclones that gave rise to a 620 by fragment were selected for furtheranalysis.

For sequencing purposes, plasmid DNA from selected clones wasPEG-purified as described in Example 34. Two microliters of PEG-purifiedplasmid DNA (approximately 100 ng) was used for cycle-sequencing withFMOL kit (Promega, Madison, Wis.) in conjunction with the PolB-5A andPolB-5B primers containing a biotin moity at the 5′ end. Biotinylationof the primers was performed using an Oligonucleotide Biotin Labelingkit (Amersham). Sequencing reactions were separated in a 8% denaturingpolyacrylamide gel, transferred to a nylon membrane and visualized asdescribed above in Example 21. The DNA sequences of the 620 by rpoB genefragment derived from the wild-type, mutant 1 and mutant 2 strains arelisted in SEQ ID NOS:135-137. The sequence of the sense strand for eachof the three TB strains is shown as the top line in SEQ ID NOS:135-137.The sequence of the anti-sense strand for the wild-type, mutant 1 andmutant 2 TB strains is listed in SEQ ID NOS:138-140, respectively.

ii) Preparation of M. tuberculosis rpoB Gene Substrates

In order to generate substrates for use in CFLP reactions, the cloned620 by fragment derived from the wild type and mutants 1 and 2 rpoB genewere amplified using the PCR. The PCRs were conducted using one primerof the primer pair labeled at the 5′ end so that the resulting PCRproduct would permit the analysis of either the sense or anti-sensestrand of the rpoB gene fragments. In order to generate substrateslabelled on the anti-sense strand, ten nanograms of plasmid DNA from thesequenced clones was used as the template in 50 μl reactions containing1 μM of each the PolB-5A primer (unlabelled) and PolB-5B primerbiotinylated at the 5′ end using Oligonucleotide Biotin Labeling kit(Amersham), 1.5 mM MgCl₂, 20 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.05% eachTWEEN-20 and NONIDET P-40, 60 μM of all 4 dNTPs and 2.5 units of Taqpolymerase. The reactions were cycled 35 times at 95° C. for 1 min, 60°C. for 1 min and 72° C. for 2 min. The resulting 620 by PCR productscontaining a biotin-labeled antisense strand were gel-purified asdescribed in Example 21. The purified fragments were dissolved in 20 μlH₂O.

To generate substrates labelled on the sense strand of the 620 byfragment of rpoB gene fragments (wild-type and mutats 1 and 2), the PCRswere conducted using 1 μM each PolB-5A primer 5′ end labeled with TMRdye using ONLY BODIPY TMR Oligonucleotide Phosphate Labeling Kit(Molecular Probes, Inc., Eugene, Oreg.) and unlabeled PolB-5B primer.The PCR reactions also contained 1.5 mM MgCl₂, 20 mM Tris-HCl, pH 8.3,50 mM KCl, 0.05% each TWEEN-20 and NONIDET P-40, 60 μM of all 4 dNTPs, 5units of Taq polymerase and 10 ng of plasmid DNA from the sequencedclones as a template in a final volume of 100 μl. The reactions werecycled for a total of 35 cycles comprising 95° C. for 1 min, 60° C. for1 min and 72° C. for 2 min.

In addition to the above PCR conditions, the PCR reactions were alsoconducted using dUTP in place of dTTP to generate uridine-containing PCRfragments. Uridine-containing PCR fragments have become the standardtype of PCR fragment analyzed in clinical laboratories. In order todemonstrate that uridine-containing PCR fragments can be used to producedistinct CFLP patterns from substrates which vary by a single base pairchange within a 620 by fragment, rpoB gene fragments containing a 5′ TMRlabel on the sense strand and uridine in place of thymidine weregenerated as follows. Uridine-containing 620 by fragments (wild-type andmutants 1 and 2) were amplified according to the PCR protocol describedabove for the generation of fragments labelled at the 5′ end of thesense strand with TMR with the exception that 2.5 mM MgCl₂ was used inplace of 1.5 mM MgCl₂ and 100 μM dATP, 100 μM dCTP, 100 μM dGTP and 200μM dUTP were used in place of the mixture containing 60 μM each of all 4dNTPs (i.e., dATP, dCTP, dGTP and dTTP).

The 620 by PCR products containing a TMR-labeled sense strand (eitheruridine- or thymidine-containing) were purified in 6% denaturing gel asdescribed above, eluted overnight, precipitated with ethanol andredissolved in 20 μl H₂O as described in Example 21, for a concentrationof approximately 15 fmoles/μl.

iii) Cleavage Reaction Conditions

Cleavage reaction conditions for analysis of the 620 by rpoB fragmentscontaining a biotin-labelled antisense strand were as follows. Sixmicroliters of biotin labeled PCR product were combined with 1 μl of10×CFLP buffer (100 mM MOPS, pH 7.5, 0.5% each TWEEN 20 and NP-40) and25 ng CLEAVASE BN enzyme. Prior to the initiation of the cleavagereaction, the DNA mixtures were denatured by incubation at 95° C. for 10sec. The reactions were then cooled to 60° C. and reaction was startedby the addition of 1 μl of 2 mM MnCl₂. The cleavage reactions wereconducted at 60° C. for 2 min. Cleavage reactions were stopped after 2min. by adding 5 μl of STOP solution (95% formamide, 10 mM EDTA and0.02% each bromphenol blue and xylene cyanol). Six microliters of eachsample were resolved by electrophoresis on a 6% denaturingpolyacrylamide gel and labeled fragments were visualized as described inExample 21. The resulting autoradiogram is shown in FIG. 84.

In FIG. 84, the lane marked “M” contains biotinylated molecular weightmarkers obtained from Amersham (Arlington Heights, Ill.) and includebands corresponding to lengths of 200, 300, 400, 500 nucleotides. Thesize of the markers and of the uncleaved rpoB substrates (620) isindicated on the left-hand side of the autoradiograph using largearrowheads. Lanes 1-3 contain the reaction products generated by thecleavage of the mutant 1, wild-type and mutant 2 substrates labelled onthe anti-sense strand, respectively. The distance of the point mutation(relative to the wild-type sequence) from the 5′ end label was 511nucleotides for the mutant 1 substrate and 499 nucleotides for themutant 2 substrate.

The results shown in FIG. 84 demonstrate that similar, but distinctlydifferent patterns of cleavage were generated from the each of the rpoBsubstrates labelled on the anti-sense strand. In comparision with thecleavage pattern generated by the wild-type substrate, the patterngenerated by cleavage of the mutant 1 substrate shows a disappearance ofBand A. A comparision of the pattern generated by cleavage of thewild-type and mutant 2 substrates shows that the mutant 2 substrate hasa significant reduction of insensity of Band B. Thus, the two mutantscan be distinguished from the wild-type and from each other.

Cleavage reaction conditions for analysis of the 620 by rpoB fragmentscontaining a TMR-labelled sense strand were as follows. Four microlitersof TMR-labeled PCR product were cleaved as described above. Cleavagereactions were stopped after 2 min. by adding 5 μl 95% formamide, 10 mMEDTA and 0.02% of methyl violet (Sigma).

The reactions were heated to 85° C. for 2 min. and five microliters ofeach reaction mixture were resolved by electrophoresis through a 12%denaturing polyacrylamide gel (19:1 cross link) with 7M urea in a buffercontaining 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The gel was run at33W (watts) for 1.5 hours. The labeled reaction products were visualizedusing the FMBIO-100 Image Analyzer (Hitachi) with the resultingfluoroimager scan shown in FIG. 85, PAnel A. the gel was thenelectrophoresed for another 1 hour, and the second scan is shown inPanel B.

In FIG. 85, two panels, A and B, are shown. Panel B represents a scan ofthe same gel shown in Panel A following a longer period ofelectrophoresis than that shown in Panel A. Thus, Panel B serves tospread out the banding pattern seen in the upper portion of Panel A(lines connecting Panels A and B show the region of expansion). In FIG.85, Panels A and B, lanes 1-4 contain the reaction products produced bycleavage of thymidine-containing substrates having a TMR-label on thesense strand derived from the mutant 1, wild-type, mutant 2 and amixture of the wild-type and mutant 2 substrates, respectively. Lane 5of Panels A and B contains the 157 by fragment derived from exon 4 ofthe tyrosinase gene (SEQ ID NO:40) labeled with TET as a marker. Lanes6-9 of Panels A and B contain the reaction products produced by cleavageof uridine-containing substrates having a TMR-label on the sense strandderived from the mutant 1, wild-type, mutant 2 and a mixture of thewild-type and mutant 2 substrates, respectively. Mixtures of thewild-type and mutant 2 substrates (lanes 4 and 9) were generated bymixing together 5 μl of each substrate after the cleavage reaction; 6 μlof the mixture was then loaded on the gel. The distance of the pointmutation (relative to the wild-type sequence) from the 5′ end label was100 nucleotides for the mutant 1 substrate and 116 nucleotides for themutant 2 substrate.

The results shown in FIG. 85 demonstrate that similar, but distinctlydifferent patterns of cleavage were generated from the each of the rpoBsubstrates labelled on the sense strand. The left hand set of each panelcontains CFLP patterns generated from PCR products containing dNTPs,while the right hand side contains CFLP patterns generated from PCRproducts in which dUTP was substituted for dTTP. Comparision of the CFLPpatterns generated from dNTP-containing amplicons of mutant 1 andwild-type reveals a marked reduction in intensity of a bandapproximately 80 nt from the labeled 5′ end (band A), in the vicinity ofthe sequence change in this mutant (100 by from the labeled 5′ end). Inaddition, a band migrating at approximately 200-250 nt from the labeled5′ end (band B) is missing in mutant 1. In contrast, comparision of thepatterns generated from wild-type and mutant 2 reveals the loss of aband 120 nt from the labeled 5′ end (band C). Furthermore, examinationof the region of the gel corresponding to 120 nt shows, particularly inPanel B, that band D is shifted downward in mutant 2 relative towild-type. In Panel B, another band, migrating just above band D(labeled band D′) also appears to be shifted downward in mutant 2relative to wild-type. Lane 4 of each panel, in which aliquots from thewild-type and mutant CFLP reactions were mixed prior to electrophoresisdemonstrates that this shift (in band D′) in mutant 2 is real and notdue to an electrophoresis artifact.

Examination of the CFLP patterns generated from the dUTP-containingamplicons demonstrates that the ability to distinguish these mutantsfrom one another, as well as from the wt, is not adversely affected bysubstitution of dUTP for dTTP and may, in fact, be enhanced. In thisexample, both mutants 1 and 2 are more readily distinguished from the wtwhen the patterns are generated from amplicons containing dUTP thandTTP. In the right-hand portion of panel A, comparison of the lanescontaining mutant 1 and wt reveals several distinctive differencesbetween the two amplicons, while others are new and unanticipated.Specifically, band A is reduced in intensity in the mutant, as comparedto the wt, in much the same way that it is in the left-hand portion ofthis panel. A band migrating at approximately 110 nt (band E) appears tobe missing from the mutant, as does a band at approximately 250 nt(compare to band B in the left-hand portion of the gel). In addition,the strong band labeled F, while not noticeably different in the threesamples containing dTTP, is much stronger in the wt pattern generatedfrom dUTP-containing amplicons than it is in the mutants. Comparison ofthe patterns generated from wt and mutant 2 also reveals a number ofpronounced differences. Most notably, a band migrating at approximately60 nt appears in mutant 2 (band G), as does a complex of 2 new bandsmigrating at approximately 150 nt (band H). Interestingly, while some ofthe elements that make each of these patterns distinct from one anotherare different if dUTP is substituted for dTTP in the PCR amplification,the vast majority of the cleavage fragments are identical in the twoexperiments. This result suggests that substitution of dUTP results insubtle alterations in the single-stranded DNA substrate which may be theresult of altered stability of secondary structures or an alteredaffinity of CLEAVASE enzymes for secondary structures containingmodified nucleotides. These differences in CLEAVASE enzyme-basedrecognition of secondary structures in DNA fragments containing dUTPprovides an unexpected benefit of using this nucleotide substitution.

b) CFLP Analysis of Mutations in the KatG Gene of M. tuberculosis

i) Generation of Plasmids Containing KatG Gene Sequences

Genomic DNA isolated from wild-type M. tuberculosis or M. tuberculosisstrains containing mutations in the katG gene associated with isoniazidresistance were obtained from Dr. J. Uhl (Mayo Clinic, Rochester,Minn.). These four strains are termed wild-type, S315T, R463L andS315T;R463L (Cockerill, III et al, J. Infect. Dis. 171:240 (1995)).Strain S315T contains a G to C mutation in codon 315 of the wild-typekatG gene. Strain R463L contains a G to T mutation in codon 463 of thewild-type gene and strain S315T;R463L contains both the G to C mutationin codon 315 and the G to T mutation in codon 463.

A 620 by region of the M. tuberculosis katG gene was amplified using thePCR from DNA derived from the above four strains. The primers used toamplify the katG gene sequences were KatG904 (5′-AGCTCGTATGGCACCGGAAC-3′(SEQ ID NO:141)) and KatG1523 (5′-TTGACCTCCCACCCGACTTG-3′ (SEQ IDNO:142)); these primers amplify a 620 by region of katG gene which spansboth the S315T and R463L mutations. The PCRs were conducted in a finalreaction volume of 100 μl and contained the KatG904 and KatG1523 primersat 0.5 μM, 1.5 mM MgCl₂, 20 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.05% eachTWEEN-20 and NONIDET P-40, 60 μM of all 4 dNTPs. The reaction mixtureswere heated at 95° C. for 3 min, then amplification was started withaddition of 5 units of Taq polymerase and continued for 35 cycles at 95°C. for 1 min, 60° C. for 1 min and 72° C. for 2 min.

To clone the PCR-amplified katG fragments, 1 μl of each PCR product wasused for ligation into the linear pT7Blue T-vector (Novagen, Madison,Wis.). The ligation products were used to transform competent JM109cells and clones containing pT7Blue T-vector with an insert wereselected by white color on LB plates containing 40 μg/ml X-Gal, 40 μg/mlIPTG and 50 μg/ml ampicillin. For each of the four PCR samples, fourcolonies were picked and grown overnight in 2 ml LB media containing 50μg/ml carbenicillin. Plasmid DNA was isolated using the alkalineminiprep protocol described in Example 34.

To analyze the cloned katG fragments, 1 μl of plasmid DNA from eachclone was amplified by PCR using 100 μl reactions containing the KatG904and KatG1523 primers at 0.5 μM, 1.5 mM MgCl₂, 20 mM Tris-HCl, pH 8.3, 50mM KCl, 0.05% each TWEEN-20 and NONIDET P-40, 60 μM of all 4 dNTPs and 5units of Taq polymerase. The PCRs were cycled 35 times at 95° C. for 1min, 60° C. for 1 min and 72° C. for 2 min. PCR products were separatedby electrophoresis on a 6% native polyacrylamide gel in 0.5×TBE bufferand clones that gave rise to a 620 by fragment were selected for furtheranalysis.

For sequencing purposes, plasmid DNA from selected clones wasPEG-purified according to the protocol described in Example 34. Twomicroliters of plasmid DNA (approximately 100 ng) was used forcycle-sequencing with FMOL kit (Promega, Madison, Wis.) in conjunctionwith the KatG904 and KatG1523 primers containing a biotin moity at the5′ end. Biotinylation of the primers was performed using anOligonucleotide Biotin Labeling kit (Amersham). Sequencing reactionswere separated in a 8% denaturing polyacrylamide gel, transferred to anylon membrane and visualized as described above in Example 21. The DNAsequences of the 620 by katG gene fragments from the wild-type andmutant strains S315T, R463L and S315T;R463L are listed in SEQ IDNOS:143-146, respectively. The sequence of the sense strand for each ofthe four katG gene fragments is shown as the top line in SEQ IDNOS:143-146, respectively. The sequence of the anti-sense strand of the620 by katG gene fragments from the wild-type and mutant strains S315T,R463L and S315T;R463L is listed in SEQ ID NOS:147-150, respectively.

ii) Preparation of M. tuberculosis KatG Gene Substrates

In order to generate substrates for use in CFLP reactions, the cloned620 by fragments derived from the wild-type and S315T, R463L andS315T;R463L M. tuberculosis strains were amplified using the PCR. ThePCRs were conducted in a final reaction volume of 100 μl and contained0.5 μM each KatG904 and KatG1523 primers, 1.5 mM MgCl₂, 20 mM Tris-HCl,pH 8.3, 50 mM KCl, 0.05% each TWEEN-20 and NONIDET P-40, 60 μM of all 4dNTPs, 5 units of Taq polymerase and 10 ng of plasmid DNA from thesequenced clones as a template. The reactions were cycled 35 times at95° C. for 1 min, 60° C. for 1 min and 72° C. for 2 min.

To obtain 620 by PCR fragments of the katG gene having a biotin label onthe sense strand, and unlabeled KatG1523 primer (SEQ ID NO:142) and5′-biotinylated KatG904 primer (SEQ ID NO:141) was used in the PCR;biotinylation was achieved using the Oligonucleotide Biotin Labeling kit(Amersham). To produce the same fragments having the TMR label on theantisense strand, unlabeled KatG904 (SEQ ID NO:141) and TMR-labeledKatG1523 (SEQ ID NO:142) primers were used in the PCR. Amplified PCRproducts were purified on a 6% denaturing gel, eluted overnight,precipitated with ethanol and redissolved in 50 μl H₂O as described inExample 21.

iii) Cleavage Reaction Conditions

The cleavage reaction conditions for analysis of katG substrateslabelled on the sense strand were as follows. Five microliters of biotinlabeled PCR product were combined with 1 μl of 10×CFLP buffer (100 mMMOPS, pH 7.5, 0.5% each TWEEN 20 and NP-40) and 25 ng CLEAVASE BNenzyme. Prior to the initiation of the cleavage reaction, the DNAmixtures were denatured by incubation at 95° C. for 10 sec. Thereactions were then cooled to 50° C. and the reaction was started by theaddition of 1 μl of 2 mM MnCl₂. The cleavage reactions were incubatedfor 2 min. at 50° C. and were stopped by adding 5 μl of a solutioncontaining 95% formamide, 10 mM EDTA and 0.02% each bromphenol blue andxylene cyanol. Four and one-half microliters of each sample were run ona 10% denaturing polyacrylamide gel and labeled fragments werevisualized following transfer to a nylon membrane as described inExample 21. The resulting autoradiogram is shown in FIG. 86.

In FIG. 86, lanes marked “M” contain biotinylated molecular weightmarkers obtained from Amersham (Arlington Heights, Ill.) and includebands corresponding to lengths of 50, 100, 200, 300, 400, 500, 700, and1000 nucleotides; the size of the markers is indicated by the use oflarge arrowheads. Lanes 1-4 contain the reaction products obtained byincubating the R463L, R463L;S315T, S315T and wild-type katG substratesin the presense of CLEAVASE BN enzyme, respectively. The mutationdistance from the 5′ end label is 485 nucleotides for the R463L mutationand 41 nucleotides for the S315T mutation when the label is present onthe sense strand.

The results shown in FIG. 86 demonstrate that similar, but distinctlydifferent patterns of cleavage were generated from the wild-type andS315L mutant (seen in both the S315T and S315;R463L substrates) katGsubstrates labelled on the sense strand. Comparision of the CFLP patternfor wild-type fragment (lane 4) shows that the S315T mutation (seen inboth mutants R463L;S315T and S315T; lanes 2 and 3) results indisappearance of Band B which is located around 40 nucleotides from theend label in the wild-type substrate. The disappearance of Band Bcorrelates very well with the distance of S315T mutation from the 5′ end(41 nucleotides from the 5′ end label on the sense strand). Subsequentexperiments have demonstrated that the R463L mutant can be distinguishedfrom wild-type by a mobility shift in a band migrating at approximately500 nt from the 5′ end label on the sense strand (the band shiftsdownward in the R463L mutant), but is difficult to resolve in many gelssystems.

The cleavage reaction conditions for analysis of katG substrates labeledon the anti-sense strand were as described for the sense strand. Fourand one-half microliters of each sample were run on a 10% denaturingpolyacrylamide gel and labeled fragments were visualized using theHitachi FMBIO-100 fluoroimager as described in Example 35(a)(iii). Theresulting scan is shown in FIG. 87.

In FIG. 87, lanes marked “M” contain plasmid pUC19 DNA digested withMspI and 3′ end labeled with fluorescein ddUTP using terminaldeoxynucleotidyl transferase as described in Example 10. This markerincludes bands corresponding to lengths of 110/111, 147, 190, 242, 331,404, 489 and 501 bp. Additional marker bands of 26, 34, and 67 by arenot visible in this figure; the size of the markers is indicated by theuse of large arrowheads. Lanes 1-4 contain the reaction productsobtained by incubating the R436L, S315T;R463L, S315T, and wild-type katGsubstrates in the presense of CLEAVASE BN enzyme, respectively. Thelocation of the single base mutation from the 5′ end label is 136nucleotides for the R463L mutation and 580 nucleotides for the S315Tmutation when the label is present on the anti-sense strand.

The results shown in FIG. 87 demonstrate that wild-type can bedistinguished from mutants containing the R463L substitution on theanti-sense strand. Comparison of the lanes containing the S315T;R463Ldouble mutant or the R463L mutant by itself demonstrates that the R463Lmutation is associated with the presence of a strong band migrating atapproximately 130 nt (band A). This result, taken with that presented inFIG. 86, demonstrates that all three of these mutants can bedistinguished from one another, as well as from wild type, by CFLPanalysis.

The CFLP technology offers cost benefits by reducing gel electrophoresisprocessing time from 12-18 hours down to 5 to 10 minutes. Adapting thereadout to multi-lane Fluorescence Image Detectors allows for anexpanded volume of work by allowing simultaneous processing of up to 48reactions. The consequent decrease in turnaround time in performing theanalyses reduces the turnaround time of reporting patient results fromdays to hours, or, as in the case of MDR-TB patients, from weeks tohours. Early detection of MDR-TB can save thousands of dollars perpatient by reducing the expense of extended stays in isolation wards,spent while testing various antibiotic treatments for efficacy.

Example 36 Rapid Identification of Bacterial Strains by CFLP Analysis

The results shown above demonstrated that CFLP™ analysis can be used todetect the presence of wild-type and drug-resistant mutations of M.tuberculosis by examining portions of gene associated with drugresistance (e.g., rpoB and katG). In order to examine whether the CFLPanalysis could be used as a method of detecting and identifying a widevariety of microorganisms, CFLP analysis was conducted using substratesderived from bacterial 16S rRNA genes.

Bacterial 16S rRNA genes vary throughout the phylogenetic tree; thesegenes do contain segments which are conserved at the species, genus orkingdom level. These features have been exploited to generate primerscontaining concensus sequences which flank regions of variability. Theseprimers have been used to amplify segments of bacterial 16S rRNA geneswhich are then characterized by either Southern blot hybridization(Greisen et al., J. Clin. Microbiol. 32:335 (1994)) or SSCP analysis(Widjojoatmondjo et al., J. Clin. Microbiol. 32:3002 (1994)). Thesetypes of analysis, while faster than traditional culturing methods, areat best limited to the differentiation of species within a particulargenus and higher bacterial taxons. However, it is often desirable todifferentiate between different strains of the same species. Forexample, a given species may contain subspecies which comprise harmlessas well as pathogenic organisms. In order to develop a technique whichwould allow the differentiation between species and/or subspecies, CFLPanalysis was applied to segments derived from bacterial 16S rRNA genes.

a) Bacterial Strains

Table 3 below lists the bacterial strains used in this study. Thesestrains were derived from the ATCC strains listed below with theexception of Desulfurococcus amylolyticus Strain Z-533 which was derivedfrom a deposit obtained from the Deutsche Sammlung von Mikroorganismen(DSM).

TABLE 3 ORGANISM STRAIN NO. CHARACTERISTICS E. coli ATCC 11303 Strain BE. coli ATCC 14948 Derived from E. coli strain K-12 E. coli SerotypeO157: H7 ATCC 43895 Produces Shiga-like toxins I and II Campylobacterjejuni subsp. ATCC 33291 Isolated from human stool jejuni Shigelladysenteriae ATCC 29027 Isolated from human stool Serotype 2 Salmonellacholeraesuis ATCC 6539 Used for germicide testing subsp. choleraesuisSerotype typhi Staphylococcus aureus ATCC 33591 Methicillin-resistantsubsp. aureus S. aureus subsp. aureus ATCC 33592 Gentamicin- andmethicillin-resistant S. aureus subsp. aureus ATCC 13565 Producesenterotoxin A and large amounts of beta-hemolysin Staphylococcus hominisATCC 29885 Methicillin control for MIC testing Staphylococcus warneriATCC 17917 Used for soap germicide testing Desulfurococcus STRAIN 3822Thermophilic archaebacterium amylolyticus

The strains listed in Table 3 represent pathogenic microorganisms withthe exception of E. coli strains B and K-12 and Desulfurococcusamylolyticus. Desulfurococcus amylolyticus was included in this study todetermine whether the concensus primers, whose design was based uponknown rRNA gene sequences, could also be used to amplify rRNA genefragments sequences from archeabacterial species whose rRNA genesequences have not been reported.

The strains listed in Table 3 were selected to provide representativesfrom several different genera (e.g., Escherichia, Shigella, Salmonella,Campylobacter, etc.) as well as to provide several representatives ofdifferent species (or subspecies) within a given genus. For example,three different strains of E. coli were chosen so that the consistency(or lack thereof) of the CFLP banding pattern generated by cleavage ofan rRNA gene substrate could be examined between species within a givengenus. In addition, E. coli Serotype O157:H7 was examined as this strainhas been implicated in hemorragic colitis outbreaks. It was of interestto examine whether the CFLP pattern observed from clevage of a rRNA genesubstrate from E. coli strains B or K-12 differed from that produced bycleavage of a rRNA gene substrate from E. coli Serotype O157:H7.

Table 4 below describes the phylogenic relationship between the strainsused in this example.

TABLE 4 Phylogenetic position of strains from Prokaryotic Small SubUnitrRNA Taxonomic List¹ 1 ARCHAEA 1.2 CRENARCHAEOTA 1.2.1CRENARCHAEOTA-GROUP-I Desulfurococcus amylolyticus 2 BACTERIA 2.13PURPLE-BACTERIA 2.13.3 GAMMA-SUBDIVISION 2.13.3.15 ENTERICS ANDRELATIVES 2.13.3.15.2 ESCHERICHIA-SALMONELLA ASSEMBLAGE Escherichia coliStrain B Escherichia coli Strain K-12-derived Escherichia coli SerotypeO157: H7 Shigella dysenteriae Serotype 2 Salmonella choleraesuis subsp.choleraesuis Serotype typhi 2.13.5 EPSILON-SUBDIVISION 2.13.5.2CAMPYLOBACTER AND RELATIVES Campylobacter jejuni subsp. jejuni 2.15GRAM-POSITIVE PHYLUM 2.15.5 BACILLUS-LACTOBACILLUS-STREPTOCOCCUSSUBDIVISION 2.15.5.10 STAPHYLOCOCCUS GROUP 2.15.5.10.2 STAPHYLOCOCCUSSUBGROUP Staphylococcus aureus subsp. aureus ATCC 33591 Staphylococcusaureus subsp. aureus ATCC 33592 Staphylococcus aureus subsp. aureus ATCC13565 Staphylococcus hominis Staphylococcus warneri ¹Data derived fromthe Ribosomal Database Project; available on the Inernet athttp://rdp.life.uiuc.edu/index.html; Maidak et al, Nucleic Acids Res.,22:3485 (1994).

b) Growth of Microorganisms

In order to minimise handling of the pathogenic strains, themicroorganisms were grown on slant cultures or on plates rather than inliquid culture.

i) Growth of Escherichia, Shigella, Salmonella, and StaphylococcusSpecies

All strains were derived from the ATCC strains listed above in Table 3as follows. A loopful of a culture previously frozen in Trypticase SoyBroth and 15% glycerol (Remel Corp., Lenexa, Kans. Cat. 06-5024) wassubcultured onto a trypticase soy agar slant (Remel, Cat. 06-4860). Thecultures were incubated overnight at 37° C.

ii) Growth of Campylobacter Species

A loopful of a culture previously frozen in Trypticase Soy Broth and 15%glycerol (Remel Corp., Lenexa, Kans. Cat. 06-5024) was subcultured ontoCampylobacter Agar supplemented with 10% sheep blood, amphotericin B,cephalothin, trimethoprim, vancomycin, and polymyxin B (BBL, Cat.21727). Inoculated plates were sealed in Campy microaerophilic pouches(BBL, Cat. 4360656) and incubated at 42° C. for 3 days.

c) Extraction of Genomic DNA from Microorganisms

For each bacterial sample, 300 μl of TE buffer (10 mM Tris-HCl l (pH 8.0at 25° C.), 1 mM EDTA) and 300 μl phenol:cloroform:isoamyl alcohol(25:24:1) were placed in a 1.5 ml microfuge tube. This combination isreferred to as the extraction buffer. A loopful (approximately 0.1 ml)of the desired bacterial strain was removed from a slant culture orplate and combined with the extraction buffer in a 1.5 ml microfuge tubeand the contents were vortexed for two minutes. The extracted DNApresent in the aqueous phase was processed for further purification asdescribed below.

Samples of E. coli and C. jejuni strains were ethanol precipitated anddissolved in 50 μl TE buffer. The samples were then treated with 0.5 μgRNase A at 37° C. for 30 min. DNA was precipitated with ethanol,collected by centrifugation and dissolved in 200 μl 10 mM Tris-HCl (pH8.0 at 25° C.).

Samples of Shigella, Salmonella, and Staphylococcus strains wereconcentrated using a MICROCON 30 filter (Amicon) to 50 μl and thentransferred to TE buffer using MICROSPIN S-200 HR gel filtration columns(Pharmacia Biotech). The samples were then treated with 0.5 μg RNase Aat 37° C. for 80 min. DNA was precipitated with ethanol, collected bycentrifugation and dissolved in 200 μl 10 mM Tris-HCl (pH 8.0 at 25°C.).

Genomic DNA of E. coli Strain B (ATCC 11303) was obtained from PharmaciaBiotech (Piscataway, N.J.; Cat. 27-4566-01, Lot 411-456601). The DNA wasdissolved in 10 mM Tris-HCl (pH 8.0 at 25° C.).

Genomic DNA from Desulfurococcus amylolyticus Strain Z-533 (DSM 3822)was isolated and purified using the standard technique of cesiumchloride centrifugation. (Bonch-Osmolovskaya, et al., Microbiology(Engl. Transl. of Mikrobiologiya) 57:78 (1988)).

The concentration of the genomic DNA preparations was determined bymeasuring the OD₂₆₀ of the preparations.

d) Design of Primer for the Amplification of 16s rRNA Genes of BacterialSpecies

Primers and probes have been reported which allow the amplification ordetection of 16S rRNA sequences from a wide variety of bacterialstrains. These oligonucleotide primers or probes represent consensussequences derived from a comparision of the 16s rRNA gene sequences froma variety of eubacterial species. For example, oligonucleotide primerssuitable for either PCR amplification or dot blot hybridization ofbacterial rRNA gene sequences have been reported (e.g., PCT PublicationWO 90/15157; Widjojoatmodjo et al., J. Clin. Microbiol. 32:3002 (1994)).Typically the conserved primer sequences are designed to flanknonconserved regions of the 16s rRNA gene with species-specificsequences.

A number of previously published conscensus primers derived from 16SrRNA gene sequences were examined for the ability to produce substratesfor use in CFLP reactions. Primers 1638, 1659 and 1743 were described inPCT Publication WO 90/15157. Primer ER10 was described in Widjojoatmodjoet al., supra. Primers SB-1, SB-3 and SB-4 represent new primers (i.e.,not previously published). The primers used in this example are listedin Table 5 below.

TABLE 5 Primers for PCR Amplification of 16S rRNA Genes SEQ ID PRIMERNO: SEQUENCE 1638 151 5′-AGAGTTTGATCCTGGCTCAG-3′ ER10 1525′-GGCGGACGGGTGAGTAA-3′ 1659 153 5′-CTGCTGCCTCCCGTAGGAGT-3′ SB-4 1545′-ATGACGTCAAGTCATCATGGCCCTTACGA-3′ 1743 1555′-GTACAAGGCCCGGGAACGTATTCACCG-3′ SB-1 156 5′-GCAACGAGCGCAACCC-3′ SB-3157 5′-ATGACGTCAAGTCATCATGGCCCTTA-3′

The oligonucleotide primers were obtained from Integrated DNATechnologies, Inc. The oligonucleotides were dissolved in 10 mM Tris-HCl(pH 8 at 25° C.) at a concentration of 20 μM. Two sets of primers weresynthesized; one set having an OH group at the 5′ end (i.e., unlabelledprimers) and the other set having the fluorescent dye TET(tetrachlorinated analog of 6-carboxyfluorescein, Applied Biosystems) atthe 5′ end (i.e., TET-labelled primers). TET-labelled primers areindicated by the use of “TET” as a suffix to the primer name (forexample, TET-1638 indicates the 1638 primer having a 5′ TET label).

The location of each of the primers listed in Table 5 is shown along thesequence of the E. coli rrsE gene (encodes a 16S rRNA) in FIG. 88. InFIG. 88 the primer sequences are shown in bold type and underlining isused to indicate complete identity between primer sequences and E. colirrsE gene sequences. The sequence of the E. coli rrsE gene is listed inSEQ ID NO:158. As shown in FIG. 88, the 1638, ER10, SB-1, SB-3, SB-4primers correspond to sequences present on the sense strand of the 16SrRNA gene. The 1659, 1743 primers correspond to sequences present on theanti-sense strand of the 16S rRNA gene.

FIG. 89 provides an alignment of the E. coli rrsE gene (SEQ ID NO:158),the Cam.jejun5 gene (a rRNA gene from C. jejuni) (SEQ ID NO:159) and theStp.aureus gene (a rRNA gene from S. aureus) (SEQ ID NO:160). Thelocation of the 1638, ER10, 1659 (shown as the complement of 1659),SB-1, SB-3, SB-4 and 1743 (shown as the complement of 1743) primers isindicated by the bold type. Gaps (dashes) are introduced to maximizealignment between the rRNA genes.

In procaryotes the ribosomal RNA genes are present in 2 to 10 copies,with an average of 7 copies in Escherichia strains. Any PCRamplification produces a mixed population of these genes and is inessence a “multiplex” PCR from that strain. The CFLP pattern representsa composite pattern from the slightly varied rRNA genes within thatorganism so no one particular rRNA sequence is directly responsible forthe entire “bar code.” In some cases these minor variations (betweenrRNA genes; see, for example, minor variations between the E. coli rRNAgens in FIG. 88) cause shifts in the minor (lower signal) bands in theCFLP pattern, allowing discrimination between very closely relatedorganisms. More dramatic sequence variations, found in most or allcopies of these genes, are seen when more distantly related organismsare compared (see, for example, the extensive variations between the E.coli, C. jejuni and S. aureus rRNA genes in FIG. 89) and these largerdifferences are reflected in the CFLP patterns as more dramatic patternchanges. Despite the variable nature of these genes, the amplificationby PCR can be performed between conserved regions of the rRNA genes, soprior knowledge of the entire collection of rRNA sequences for anymicrobe of interest is not required.

Three primers (TET-1638, TET-ER10, and TET-SB-4) were used for makingthe 5′ end fluorescently labeled fragments of the sense strand of 16SrRNA genes; two other primers (TET-1659 and TET-1743) were used formaking labeled fragments of the antisense strands.

The predicted size of PCR products produced by amplification of 16s rRNAgene sequences from a variety of bacterial genera using the indicatedprimer pairs is shown in Table 6. In Table 6, the size of the predictedPCR product is based upon the known sequence of the 16S rRNA gene in theindicated species. The following abbreviations are used in Table 6: Dco(Desulfurococcus); E.co (E. coli), Cam (Campylobacter) and Stp(Staphylococus). The location of the PCR product relative to thesequence of the E. coli rrsE gene (see FIG. 88) is given in the lastcolumn.

TABLE 6 Combinations of Primers for PCR Amplification of 16S rRNASequences Primer Sense Anti-Sense Labeled Size (bp) Position Pair PrimerPrimer Strand Dco E. co Cam Stp (E. co) A TET-1638 1659 sense 350 348347  8-357 B TET-1638 1743 sense 1388 1365 1397   8-1395 C TET-ER10 1659sense 254 254 263 104-357 D TET-ER10 1743 sense 1278 1292 1271 1303 104-1395 E 1638 TET-1659 antisense 350 348 347  8-357 F ER10 TET-1659antisense 254 254 263 104-357 G TET-SB-4 1743 sense 208 208 1188-1395 HTET-1743 1638 antisense 1388 1365 1397   8-1395 I TET-1743 ER10antisense 1278 1292 1271 1303  104-1395 J SB-4 TET-1743 antisense 208208 1188-1395 K SB-1 TET-1743 antisense 305 297 296 296 1099-1395 L SB-3TET-1743 antisense 208 208 208 1188-1395

e) PCR Amplification of 16S rRNA Gene Sequences

The ability of each primer pair listed in Table 6 to amplify 16S rRNAgene sequences from each bacterial strain listed in Table 3 wasexamined. It is well known that commerical preparations of recombinantTaq DNA polymerase contain various amount of E. coli 16S rRNA genesequences. In order to minimize amplification of contaminating E. coli16S rRNA sequences during the amplification of bacterial DNA samples,AMPLITAQ DNA polymerase, LD (Low DNA) (Perkin Elmer) was used in thePCRs. This preparation of Taq DNA polymerase is tested by themanufacturer to verify that less than or equal to 10 copies of bacterial16S ribosomal RNA gene sequences are present in a standard 2.5 unitaliquot of enzyme.

Each primer pair (Table 6) was tested in PCRs. The PCR reactionscontained 10 mM Tris-HCl (pH 8.3 at 25° C.), 50 mM KCl, 1.5 mM MgCl₂,0.001% w/v gelatin, 60 μM each of dGTP, dATP, dTTP, and dCTP, 1 μM eachof one 5′-TET labeled and one unlabeled primers, 2.5 units AMPLITAQ DNApolymerase, LD. The reactions were conducted in a final volume of 50 μlusing AMPLITAQ DNA polymerase, LD, Lot E0332, or 100 μl volume usingAMPLITAQ DNA polymerase, LD, Lot D0008. The amount of genomic DNA addedvaried from 6 to 900 ng per PCR. Control reactions which contained noinput bacterial genomic DNA were also run to examine the amount of 16SrRNA product produced due to contaminats in the AMPLITAQ DNA polymerase,LD preparations.

PCR reactions were performed on PTC-100 Programmable Thermal Controller(MJ Research, Inc.). Two sets of cycling conditions were utilized. Thefirst set of conditions comprised 30 cycles of 95° C. for 30 sec; 60° C.for 1 min; 72° C. for 30 sec; after the last cycle the tubes were cooledto 4° C. The second set of conditions comprised 30 cycles of 95° for 30sec; 60° C. for 1 min; 72° C. for 90 sec; after the last cycle the tubeswere cooled to 4° C. Thus, the difference between the two cyclingconditions is the length of time the reactions are held at theelongation temperature (72° C.). These two elongation times were testedbecause the predicted size of the 16S rRNA targets varied from 208 to1388 by depending on the primer pair used in the amplification.

As a rule of thumb, when the target to be amplified is less than 500 byin length, a 30 sec elongation step is used; when the target is about500-1000 by in length, an elongation step of 30 to 60 sec is used; whenthe target is greater than 1 kb in length, the elongation is conductedfor approximately 1 min per 1 kb length. While the first set of PCRconditions (30 sec elongation step) worked with the longer amplicons,the yield was lower than that obtained when the second set of PCRconditions (90 sec elongation) was used.

Following the thermal cycling, 400 μl of formamide containing 1 mM EDTAwas added to each sample and the samples were concentrated to a volumeof 40 μl in a MICROCON 30. The samples (40 μl) were loaded on adenaturing 6% polyacrylamide gel (7 M urea, 0.5×TBE running buffer),that was prewarmed to 50-55° C. prior to the loading of the samples. Thesamples were run at 20 W for 20 min (200-350 by fragments) or 40 min(more than 1 kb fragments). The gels were scanned using a FluorescentMethod Bio Image Analyzer Model 100 (FMBIO-100, Hitachi) with a 585 or505 nm filter.

The results of these PCRs showed that each primer pair (Table 6) testedsuccessfully amplified a fragment of the expected size. Thus the primerpairs shown in Table 6 are suitable for the amplification of end labeledDNA fragments using genomic DNA from variety of prokaryotes includingarchaea, gram-positive and gram-negative bacteria, different species ofthe same genus and different strains of the same species. These PCRsalso demonstrated that, although the amount of genomic DNA present inthe PCR varied from strain to strain, the yield of the amplified productwas always many-fold higher than the trace yield of product from the E.coli genomic DNA present in AMPLITAQ DNA polymerase, LD, seen in thereactions which contained no input bacterial genomic DNA.

f) Preparation of 16S rRNA Gene Substrates

To generate labelled PCR products corresponding to bacterial 16S rRNAsequences for use in CFLP reactions, the following primer pairs wereused in PCRs.

1. The SB-1/TET-1743 pair was used to amplify an approximately 297 byfragment from genomic DNA derived from Desulfurococcus amylolyticus (DSM3822), E. coli Strain K-12 (ATCC 14948), S. aureus subsp. aureus (ATCC33591) and S. aureus subsp. aureus (ATCC 33592). The resulting PCRproduct contains a 5′ TET-label on the antisense strand.

2. The TET-SB-4/1743 pair was used to amplify an approximately 208 byfragment from genomic DNA derived from E. coli Stain B (ATCC 11303), E.coli Strain K-12 (ATCC 14948), E. coli Serotype 0157: H7 (ATCC 43895),Shigella dysenteriae Serotype 2 (ATCC 29027), and Salmonellacholeraesuis subsp. choleraesuis Serotype typhi (ATCC 6539). Theresulting PCR product contains a 5′ TET-label on the sense strand.

3. The 1638/TET-1659 pair was used to amplify an approximately 350 byfragment from genomic DNA derived from E. coli Stain B (ATCC 11303), E.coli Strain K-12 (ATCC 14948), E. coli Serotype 0157: H7 (ATCC 43895),Shigella dysenteriae Serotype 2 (ATCC 29027), and Salmonellacholeraesuis subsp. choleraesuis Serotype typhi (ATCC 6539). Theresulting PCR product contains a 5′ TET-label on the antisense strand.

4. The TET-ER10/1743 pair was used to amplify an approximately 1292 byfragment from genomic DNA derived from E. coli Strain K-12 (ATCC 14948)and Campylobacter jejuni subsp. jejuni (ATCC 33291). The resulting PCRproduct contains a 5′ TET-label on the sense strand.

5. The 1638/TET-1659 pair was used to amplify an approximately 350 byfragment from genomic DNA derived from E. coli Serotype 0157: H7 (ATCC43895), Salmonella choleraesuis subsp. choleraesuis Serotype typhi (ATCC6539), Shigella dysenteriae Serotype 2 (ATCC 29027), S. aureus subsp.aureus (ATCC 33591), S. aureus subsp. aureus (ATCC 33592), S. aureussubsp. aureus (ATCC 13565), S. hominis (ATCC 29885), and S. warneri(ATCC 17917).

The PCRs were conducted as described in section (e) above. Two separatePCR reactions were performed using 0.2 μg of genomic DNA derived fromCamylobacter jejuni subsp. jejuni (ATCC 33291) and the TET-ER10/1743primer pair. One reaction was conducted in a final volume of 50 μl andused an extension step of 30 sec at 72° C. during thermal cycling. Thesecond reaction was conducted in a final volume of 100 μl and used anextension step of 90 sec at 72° C. The yield of PCR product produced inthe second reaction was 76% higher (as compared to first reaction).

Following the amplification reaction, the samples were processed forelectrophoresis on denaturing polyacrylamide gels as described insection (e) above. After electrophoresis, the desired bands were cutfrom the gel and eluted by placing the gel slice into 0.4 ml of asolution containing 0.5 M ammonium acetate, 0.1 mM EDTA and 0.1% SDS.The mixture was then incubated at 55° C. for 2 h and then at 37° C. for12 h. The samples were concentrated to 25 μl using a MICROCON 30(Amicon) and transferred into water using S-200 MICROSPIN columns(Pharmacia).

g) Cleavage Reaction Conditions

Cleavage reactions were conducted in a final volume of 10 μl volumecontaining approximately 0.2 to 1 pmole (as indicated below) 5′TET-labeled DNA substrate, 10 ng CLEAVASE BN enzyme (Third WaveTechnologies), 1×CFLP buffer and 0.2 mM MnCl₂. The reactions were firstassembled as a 9 μl mixture lacking MnCl₂; this mixture was heated to95° C. for 10 sec and then cooled down to the desired incubationtemperature (45° C., 50° C. or 65° C.). Optimal reaction temperature foreach substrate was chosen based on even distribution of bands, and thepresence of some undigested material to indicate representation ofmolecules all the way up to full length. Selected optimal temperaturesfor each substrate are indicated in the description of FIGS. 90-93below.

The cleavage reaction was started by the addition of 1 μl of 2 mM MnCl₂.Following incubation at the desired temperature for 2 min, the reactionwas stopped by the addition of 10 μl of a solution containing 95%formamide, 5 mM EDTA, 5% glycerol and 0.02% methyl violet. Uncut or “noenzyme” controls were set up for each substrate as described above withthe exception that H2O was used in place of the CLEAVASE BN enzyme.Samples (approximately 4 to 8 μl) were run on 6 to 12% denaturingpolyacrylamide gels (19:1 cross link) with 7 M urea in a buffercontaining 45 mM Tris Borate, pH 8.3, 1.4 mM EDTA at 15 to 20 W for 9minutes (specific gel percentages are indicated below in thedescriptions of FIGS. 90-93). The gels were then scanned using aFMBIO-100 (Hitachi) with the 585 nm filter.

The resulting fluoroimager scans are shown in FIGS. 90-93. In FIG. 90,the cleavage products generated by cleavage of an approximately 297 by16S rRNA substrate generated using the SB-1/TET-1743 pair and genomicDNA derived from Desulfurococcus amylolyticus (DSM 3822), E. coli StrainK-12 (ATCC 14948), S. aureus subsp. aureus (ATCC 33591) and S. aureussubsp. aureus (ATCC 33592) is shown. Lanes 1-4 contain the productsgenerated by incubation of the substrate derived from Desulfurococcusamylolyticus (DSM 3822), E. coli Strain K-12 (ATCC 14948), S. aureussubsp. aureus (ATCC 33591) and S. aureus subsp. aureus (ATCC 33592) inthe absense of CLEAVASE BN enzyme, respectively. Lanes 5-8 contain theproducts generated by incubation of the substrate derived fromDesulfurococcus amylolyticus (DSM 3822), E. coli Strain K-12 (ATCC14948), S. aureus subsp. aureus (ATCC 33591) and S. aureus subsp. aureus(ATCC 33592) in the presence of CLEAVASE BN enzyme, respectively. TheCFLP reactions were performed using approximately 1 pmole of each PCRproduct and the cleavage reactions were incubated at 50° C. for 2 min.The cleavage products were resolved by electrophoresis on an 8%polyacrylamide gel, as described above.

The results shown in FIG. 90 demonstrate that distinct CFLP patterns areobtained using the Desulfurococcus amylolyticus (DSM 3822), E. coliStrain K-12 (ATCC 14948) and S. aureus subsp. aureus substrates. Thesame CFLP pattern was generated by cleavage of the two S. aureus subsp.aureus substrates (lanes 7 and 8); these two S. aureus subsp. aureusstrains (ATCC 33591 and 33592) are considered different subspecies basedupon differences in sesitivities to the antibiotics methicillin andgentamicin. Resistant or sensitivity to these antibiotics is notassociated with mutation in the 16S rRNA gene; therefore it was notexpected that different CFLP patterns would be observed using a 16S rRNAsubstrate.

The results shown in FIG. 90 show that the SB-1/TET-1743 pair can beused to generate substrates for CFLP analysis which allow theidentification and discrimination of Desulfurococcus amylolyticus (DSM3822), E. coli Strain K-12 (ATCC 14948) and S. aureus subsp. aureus.

In FIG. 91, Panel A shows the reaction products generated by cleavage ofan approximately 208 by 16S rRNA substrate generated using theTET-SB-4/1743 pair and genomic DNA derived from E. coli Stain B (ATCC11303), E. coli Strain K-12 (ATCC 14948), E. coli Serotype 0157: H7(ATCC 43895), Shigella dysenteriae Serotype 2 (ATCC 29027), andSalmonella choleraesuis subsp. choleraesuis Serotype typhi (ATCC 6539).The TET-SB-4/1743 pair amplifies a portion of the 16S rRNA gene locatedin the 3′ region of the gene (see FIG. 88).

The CFLP reactions shown in FIG. 91, Panel A were performed usingapproximately 0.7 pmole of each PCR product and the cleavage reactionswere incubated at 50° C. for 2 min. The cleavage products were resolvedby electrophoresis on an 8% denaturing polyacrylamide gel, as describedfor FIG. 90.

In FIG. 91, Panel B shows the reaction products generated by cleavage ofan approximately 350 by 16S rRNA substrate generated using the1638/TET-1659 pair and genomic DNA derived from E. coli Stain B (ATCC11303), E. coli Strain K-12 (ATCC 14948), E. coli Serotype 0157: H7(ATCC 43895), Shigella dysenteriae Serotype 2 (ATCC 29027), andSalmonella choleraesuis subsp. choleraesuis Serotype typhi (ATCC 6539).The 1638/TET-1659 pair amplifies a portion of the 16S rRNA gene locatedin the 5′ region of the gene (see FIG. 88).

The CFLP reactions shown in FIG. 91, Panel B were performed usingapproximately 1 pmole of each PCR product and the cleavage reactionswere incubated at 45° C. The cleavage products were resolved byelectrophoresis on an 8% polyacrylamide gel.

The lanes marked “M” in FIG. 91, Panels A and B contain plasmid pUC19DNA digested with MspI and 3′ end labeled with fluorescein ddUTP usingterminal deoxynucleotidyl transferase as described in Example 10. Thismarker includes bands corresponding to lengths of 26, 34, 67, 110/111,147, 190, 242 and 331 bp. Additional marker bands of 404, 489 and 501 byare not visible in this figure. In Panel A, lanes 1-5 contain the uncut(i.e., no enzyme) controls and lanes 6-10 contain the cleavage productsgenerated by the incubation of substrates derived from E. coli Stain B(ATCC 11303), E. coli Strain K-12 (ATCC 14948), E. coli Serotype 0157:H7 (ATCC 43895), Shigella dysenteriae Serotype 2 (ATCC 29027), andSalmonella choleraesuis subsp. choleraesuis Serotype typhi (ATCC 6539),respectively. In Panel B, lanes 1-5 contain the uncut (i.e., no enzyme)controls and lanes 6-10 contain the cleavage products generated by theincubation of substrates derived from E. coli Stain K-12 (ATCC 14948),E. coli Strain B (ATCC 11303), E. coli Serotype 0157: H7 (ATCC 43895),Shigella dysenteriae Serotype 2 (ATCC 29027), and Salmonellacholeraesuis subsp. choleraesuis Serotype typhi (ATCC 6539),respectively.

The lower molecular weight materials seen in the “uncut” lanes has beenfound to be due to degradation of the gel-purified material afterstorage for several days in dH2O. This degradation may be due toenvironmental nucleases that are active when EDTA is not present in thestorage solution (i.e., the necessary metal ions may be present in traceamounts). This degradation is effectively supressed by inculsion of tRNAin the storage solution (see Example 21). The degradation seen in theseuncut controls (Pane B, lanes 1-5) does not effect the CFLP results.

The results shown in FIG. 91 demonstrate that some regions of the 16SrRNA genes are more variable than others, and that analysis of theseregions are particularly useful when comparing very closely relatedorganisms. For example, substrates generated by the 1638/TET-1659 pair(which amplifies a portion of the 16S rRNA gene located in the 5′ regionof the gene) can be used to generate CFLP patterns which distinguish notonly between the DNA derived from the genera of Escherichia, Shigella,and Salmonella (Panel B, lanes 6-10), but which also creates distinctcleavage patterns from the DNA derived from the three strains of E. colitested (i.e., strains B, K-12 and 0157: H7) (Panel B lanes 6-8).

In contrast, no substantial difference in CFLP patterns was observedbetween the strains of the Escherichia-Salmonella assemblage for DNAfragments produced using the TET-SB-4/1743 pair which generates anapproximately 208 by fragment located near the 3′ end of 16S rRNA genes(Panel A, lanes 6-10). This contrast in the degree of variation betweenthe 5′ and 3′ regions of the 16S rRNA genes is consistent with theresults reported by Widjojoatmondjo et al., supra, in which thecomparisons between strains of the Escherichia-Salmonella assemblagewere made by SSCP analysis.

Since each organism has multiple copies of the 16S rRNA gene, and theseco-amplify in each PCR, it was important to show that the products ofdifferent amplifications from the same organism produced the samecleavage pattern. In FIG. 92, the cleavage products generated bycleavage of an approximately 1292 by 16S rRNA substrate generated usingthe TET-ER10/1743 pair in two separate PCR reactions from Campylobacterjejuni subsp. jejuni (ATCC 33291) are shown in lanes 2 and 3. Forcomparision, the same region amplified from E. coli Strain K-12 (ATCC14948) is shown in lane 1. The CFLP reactions were performed usingapproximately 60 fmole of each PCR product and the cleavage reactionswere incubated at 50° C. for 2 min. Reactions were stopped by theaddition of 95% foramide, 5 mM EDTA, 5% glycerol and 0.02% methylviolet. The cleavage products were resolved by electrophoresis on a 6%denaturing polyacrylamide gel as described above.

The results shown in FIG. 92 demonstrate that very different CFLPpatterns were generated using substrates from Gamma (Escherichia,lane 1) and Epsilon (Campylobacter, lanes 2 and 3) subdivisions ofPurple bacteria, but that the same CFLP pattern was observed between theproducts of separate PCR reactions on the same genomic DNA (lanes 2 and3).

In FIG. 93, the cleavage products generated by cleavage of anapproximately 350 by 16S rRNA substrate generated using the1638/TET-1659 pair and genomic DNA derived from E. coli Serotype 0157:H7 (ATCC 43895), S. choleraesuis subsp. choleraesuis Serotype typhi(ATCC 6539), Shigella dysenteriae Serotype 2 (ATCC 29027), S. aureussubsp. aureus (ATCC 33591), S. aureus subsp. aureus (ATCC 33592), S.aureus subsp. aureus (ATCC 13565), S. hominis (ATCC 29885), and S.warneri (ATCC 17917) are shown in lanes 1-8, respectively. The CFLPreactions were performed as described above, using approximately 200fmol of each PCR product; the cleavage reactions were incubated at 65°C. for 2 min. The cleavage products were resolved by electrophoresis ona 10% denaturing polyacrylamide gel as described above.

The results shown in FIG. 93 demonstrate that very different CFLPpatterns were produced using DNA derived from strains representingPurple bacteria (lanes 1-3) and the Gram-positive phylum (lanes 4-8). Asubstantial difference between CFLP patterns was detected between thegenera Escherichia (lane 1), Salmonella (lane 2), and Shigella (lane 3).

Additionally, a substantial difference between the CFLP patterns wasdetected between species of Staphylococcus aureus (lanes 4-6), hominis(lane 7), and warneri (lane 8). No substantial difference between CFLPpatterns was observed between the three strains of Staphylococcus aureussubsp. aureus ATCC 33591 (lane 4), ATCC 33592 (lane 5), and ATCC 13565(lane 6). These S. aureus isolates differ in reported antibioticresistance, but are so closely related that the rRNA genes do not yetshow divergence by CFLP analysis.

The above results demonstrate that CFLP analysis can be used todiscriminate between bacterial genera as well as between differentspecies and subspecies (depending on the region of the 16S rRNA geneused as the substrate). A comparison of the CFLP patterns generatedwithin the same or similar genera (e.g., Salmonella, Shigella and E.coli) shows an overall similarity in the banding pattern withdifferences revealed as changes in a small subset of the bands. When thecomparision is made across different genra (e.g., between E. coli and S.aureus) a more striking change in barcode pattern is evident indicatingthat CFLP patterns may not only be used to detect differences betweenorganisms, but the degree to which the patterns change may be used toassess the degree of evoluntionary divergence between organisms.

Substrates for CFLP analysis were produced by PCR amplification usingdifferent sets of primers. Some primer pairs (sets) are reported to beuniversal for all procaryotic organisms; other primer pairs have beenobserved to be specific for representatives of lower taxons (See, PCTPublication WO 90/15157). Except for the primer sequences, no knowledgeof the DNA sequence of the rRNA gene from any specific organism(s) isrequired for amplification and CFLP analysis of bacterial 16S rRNAgenes.

Distinct CFLP patterns were observed between representatives of archeaeaand eubacteria, different phyla of eubacteria, different phyla withineubacteria, different subdivisions of the same phylum, different generaof the same assemblage, different species of the same genus anddifferent strains of the same species. Distinct signatures in CFLPpatterns were found that allowed discrimination of pathogenic isolates,including those associated with food poisoning, from innocous members ofthe normal flora.

While the PCR products generated using genomic DNA from differentorganisms with the same set of primers are indistinguishable by theirmobility during gel electrophoresis (on non-gradient polyacrylamidegels), the CLEAVASE BN enzyme cleaves these PCR products into shorterfragments thereby generating a characteristic set of cleavage products(i.e., a distinct CFLP pattern signature). The pattern of cleavageproducts generated is reproducible; DNA substrates generated inindependent PCRs from the same organism using a given primer pair yieldthe same pattern of cleavage products.

CFLP patterns can be generated using large DNA fragments (e.g., at leastabout 1.6 kb) and thus could cover the entire length of the bacterial16S rRNA gene. CFLP analysis can also be used in conjunction withshorter DNA fragments (about 200 bp) which are located at differentpositions throughout the 16S rRNA gene.

Example 37 CFLP Analysis of Substrates Containing Nucleotide Analogs

The effect of using various nucleotide analogs to generate substratesfor CFLP reactions was examined. As discussed below, nucleotide analogsare used in PCRs for several reasons; therefore, the ability to analyzethe modified products of PCRs (i.e., nucleotide analog-containing PCRproducts) by CFLP analysis was investigated. The 7-deaza purine analogs(7-deaza-dATP and 7-deaza-dGTP) serve to destabilize regions ofsecondary structure by weakening the intrastrand stacking of multipleadjacent purines. This effect can allow amplification of nucleic acidsthat, with the use of natural dNTPs, are resistant to amplificationbecause of strong secondary structure (McConlogue et al., Nucleic AcidsRes. 16:20 (1988)).

Similarly, the analog dUTP is often used to replace dTTP, but fordifferent reasons. dUTP-containing DNA (this nomencature is shorthandfor PCR products generated using dUTP; the actual PCR product willcontain dUMP) can be destroyed by the enzymatic activity of uracil DNAglycosylase (UDG) while dTTP-containing DNA is untouched. When PCRproducts are produced containing dUMP in place of dTMP, UDG can be usedin all subsequent reactions to eliminate false positive results due tocarry-over from the earlier PCRs, without preventing amplification fromthe normal DNA of interest. This method is widely used in clinicallaboratories for performing PCR and thus this method would be used bymost clinical laboratories using PCR in conjunction with CFLP analysisfor pathogen typing. Thus, the ability of the CFLP reaction to suitablycleave dUTP-containing DNA fragments (i.e., produce strong reproducibleband patterns) was examined.

For these comparisons, substrates corresponding to a 157 by fragmentderived from exon of the wild-type and R422Q mutant of the humantryosinase gene were generated by PCR amplification using either 1) thestandard mixture of dNTPs (i.e., dATP, dCTP, dGTP and dTTP); 2) dUTP inplace of dTTP; 3) 7-deaza-dGTP (d⁷GTP) in place of dGTP; and 4)7-deaza-dATP (d⁷ATP) in place of dATP. These substrates were thenincubated with CLEAVASE BN enzyme and the effect the presence of thevarious nucleotide analogs on the cleavage pattern was examined.

a) Preparation of Substrates Containing Nucleotide Analogs

A 157 by fragment of the human tyrosinase gene (exon 4) was amplified inPCRs using the following pair: 5′ CACCGTCCTCTTCAAGAAG 3′ (SEQ ID NO:42)and 5′ biotin-CTGAATCTTGTAGATAGCTA 3′ (SEQ ID NO:43). Plasmidscontaining cDNA derived from the wild-type or R422Q mutant of thetyrosinase gene were used as template (see Example 10 for a descriptionof these plasmids). The resulting double-stranded PCR products containthe 5′ biotin label on the anti-sense strand such that sequence detectedin the CFLP reaction is SEQ ID NO:48 (wild-type anti-sense strand) orSEQ ID NO:66 (R422Q mutant anti-sense strand). All PCRs were conductedin a final volume of 100 μl. dATP, dCTP, dGTP, dTTP and dUTP wereobtained from Perkin Elmer; d′ATP and d⁷GTP were obtained fromPharmacia. Taq DNA polymerase was obtained from Promega. The PCRmixtures were assembled as shown below in Table 7.

TABLE 7 Reaction Components (Stock) Aliquot (Final) Plasmid cDNA 4 ng/ul1 ul 40 pg PCR Buffer¹ 10X 10 ul 1X Unlabelled primer 100 μM 0.25 μl 25pmole Labeled primer 100 μM 0.25 μl 25 pmole dATP 10 mM 1 μl 100 μm dCTP10 mM 1 μl 100 μm dGTP 10 mM 1 μl 100 μm dTTP 10 mM 1 μl 100 μm d⁷ATP² 5mM 2 μl 100 μm d⁷GTP³ 5 mM 2 μl 100 μm dUTP⁴ 20 mM 4 μl 800 μm Taqpolymerase 5 μ/μl 0.5 μl 2.5 units dH2O to 100 μl ¹1X concentrationcontains 20 mM Tris-HCl, pH 8.5; 1.5 mM MgCl₂; 50 mM KCl; 0.5% TWEEN 20;and 0.5% NP-40. ²d⁷ATP completely substituted for dATP in the PCR.³d⁷GTP completely substituted for dGTP in the PCR. ⁴dUTP completelysubstituted for dTTP in the PCR. Other nucleotides were present at afinal concentration of 200 μm. In this reaction, the PCR buffer used wasthe 10X buffer (500 mM KCl, 100 mM Tris-Cl, pH 9.0, 1.0% TRITON X-100)provided by Promega. 25 mM MgCl₂ was added separately to a finalconcentration of 2.5 mM.

Wild-type and the mutant R422Q substrates were amplified using thenatural and substituted nucleotide analogs listed above. For reactionscontaining the natural dNTPs, d⁷ ATP and d⁷GTP, all reaction componentswere added together. Reactions containing dUTP were initially assembledwithout the polymerase (see below).

The assembled reactions were placed in a thermocylcer (MJ Research,Watertown, Mass.) that was preheated to 95° C. The tubes were allowed toincubate for one minute at 95° C. before amplification. The program wasthen set at 94° C. for 30 minutes, 50° C. for one minute, 72° C. degreesfor two minutes for 34 cycles with a final 72° C. incubation for 5minutes.

Reactions containing dUTP were performed with a “hot start.” Allcomponents except the polymerase were mixed, heated to 95° C. for 1minute, then cooled to 72° C. Taq polymerase (2.5 units) was then addedin 10 μl of 1×PCR buffer for a final volume of 100 μl.

At the end of the amplification, the PCR products were made 0.3M NaOAc,with the exception of reactions containing dUTP; the dUTP-containingreactions were brought to 2M NH₄OAc; all were then precipitated by theaddition of 2.5 volumes (total aqueous volumes) of absolute ethanol. TheDNA pellets were collected by centrifugation and then dried undervacuum. The pellets were resuspended in 10 μl of T10E0.1 buffer (10 mMTris-HCl, pH 8.0; 0.1 mM EDTA) and 10 μl of STOP solution (95%formamide, 10 mM EDTA, 0.05% each bromophenol blue and xylene cyanol)(20 μl T10E0.1 and 16 μl of STOP for the dUTP-containing reactions). Thetubes were then heated to 85° C. for 2 minutes and the mixtures wereresolved by electrophoresis through 10% (6% for dUTP) denaturingacrylamide gel (19:1 cross link) with 7M urea in a buffer of 45 mM TrisBorate, pH 8.3, 1.4 mM EDTA.

The PCR products corresponding to the 157 by substrate derived from thewild-type and R422Q mutant were gel purified as described in Example 21.The gel-purified DNAs were resuspended in T10E0.1 buffer using thefollowing volumes: 40 μl for fragments containing only dNTPs; 40 μl forfragments containing d⁷ATP; 25 μl for fragments containing d⁷GTP and 25μl for fragments containing dUTP.

b) Cleavage Reaction Conditions

The gel purified 157 by tyrosinase substrates containing naturaldeoxynucleotides and nucleotide analogs were analyzed in cleavagereactions as follows. Final reaction mixtures comprised 1 μl of theresuspended gel-purified DNA (see section (a) above) and 25 ng CLEAVASEBN enzyme in 10 mM MOPS, pH 7.5 with 0.2 mM MnCl₂, and 0.05% each TWEEN20 and NP-40 in a volume of 20 μl. No enzyme controls were assembled inwhich distilled water replaced the CLEAVASE BN enzyme. The subsuprateDNAs were distributed into reaction tubes and brought to a volume of 15μl with H2O. The remaining reaction components were mixed in a volume of5 μl (i.e., at a 4× concentration). The DNAs were heated for 15 sec. at95° C. to denature the DNA. The cleavage reactions were initiated by theaddition of 5 μl of the enzyme/buffer mixture (the 4× concentrate). Thecleavage reactions were incubated at 45° C. for three minutes, and thereactions were terminated by the addition of 16 μl of Stop solution(described in section a). Seven microliters of each sample was heated to85° C. for two minutes prior to loading onto a 10% denaturing acrylamidegel (19:1 cross linke), with 7M urea in a buffer of 45 mM Tris Borate pH8.3, 1.4 mM EDTA. The gel was run at a constant 800 V until thebromophenol blue had migrated the length of the gel.

Following electrophoresis, the biotinylated fragments were detected asdescribed in Example 10 with the exception that 4 μl of the SAAPconjugate was added to 100 ml of USB blocking buffer (1:25,000dilution). After washing, 5 mls of CDPSTAR reagent was used as thechemiluminescent substrate. The resulting autoradiogram is shown in FIG.94.

In FIG. 94, the lanes marked “M” contain biotinylated molecular weightmarkers obtained from Amersham (Arlington Heights, Ill.) and includebands corresponding to lengths of 50, 100 and 200 nucleotides (sizeindicated by use of numbers and large arrowheads). Lanes 1-8 containreaction products obtained by incubation of the substrates in theabsence of CLEAVASE BN enzyme (i.e., no enzyme or uncut controls). Lanes9-16 contain reaction products obtained by incubation of the substratesin the presence of CLEAVASE BN enzyme. Lanes 1, 3, 5, 7, 9, 11, 13 and15 contain the wild-type substrate; lanes 2, 4, 6, 8, 10, 12, 14 and 16contain the R422Q mutant substrate. The products shown in lanes 1, 2, 9and 10 were generated from substrates generated using dNTPs in the PCRs.The products shown in lanes 3, 4, 11 and 12 were generated fromsubstrates generated using dUTP in place of dTTP in the PCRs. Theproducts shown in lanes 5, 6, 13 and 14 were generated from substratesgenerated using d⁷GTP in place of dGTP in the PCRs. The products shownin lanes 7, 8, 15 and 16 were generated from substrates generated usingd⁷ ATP in place of dATP in the PCRs. It can be seen from this examplethat modified DNA fragments are suitable for cleavage in CFLP reactions.Though the banding pattern is substantially different with thesesubstitions, the wild-type and R422Q mutant DNAs are readilydistinguishable in all cases.

While not limiting the invention to any particular theory, the changesin banding patterns observed when nucleotide analogs are utilized can beattributed to two sources. In all cases, but particularly in referenceto the 7-deaza purines, the use of nucleotide analogs may substantiallychange the nature and stability of the intrastrand folded structuresformed during the cleavage reaction. As a consequence, the locations ofthe cleavage sites would naturally shift. In addition, the substitutionof the modified nucleotides may change the affinity of the cleavageenzyme for the folded cleavage structure, either strengthening orweakening cleavage at a particular site.

Examination of the variations seen between the wild-type and R422Qmutant when different analogs are used also shows that the use of thesesubstituants can enhance the contrast between the variants. For example,with regard to the cleavage products of the two substrate DNAs(generated using dUTP or dTTP) in the region just above the 50 bymarker: one significant band that reduces in intensity between thewild-type and the mutant is more dramatically reduced in thedU-containing samples.

The results shown in FIG. 94 demonstrate that nucleotide analogs may beused for the generation of CFLP substrates. The substrates derived fromthe wild-type or R422Q mutant of the tyrosinase gene which containnucleotide analogs produce distinct cleavage patterns which allow thediscrimination and identification of the mutant and wild-type alleles.

This example demonstrates that even with 100% substitution with either7-deaza-GTP for dGTP or 7-deaza-ATP for dATP, robust CFLP patterns aregenerated, although the precise sites of clevage are different in thedNTP-containing and 7-deaza-dNTP containing substrates. The aboveresults also demonstrated that single base changes present within DNAfragments containing nucleotide analogs still inflence the foldedstructure sufficiently to cause cleavage pattern changes similar tothose seen when DNA fragments lacking nucleotide analogs are analyzedusing the CFLP assay.

From the above it is clear that the invention provides reagents andmethods to permit the rapid screening of nucleic acid sequences forvariations. These methods allow the identification of viral andbacterial pathogens as well as permit the detection of mutationsassociated with gene sequences (e.g., mutations associated with multipledrug resistance in M. tuberculosis or mutations associated with humandisease). These methods provide improved means for the identificationand characterization of pathogens.

1. A composition comprising an isolated modified thermostableeubacterial DNA polymerase enzyme having 5′ nuclease activity butlacking polymerase activity, and a nucleic acid molecule.
 2. Thecomposition of claim 1, further comprising a target nucleic acid,wherein at least a portion of said nucleic acid molecule iscomplementary to at least a portion of said target nucleic acid.
 3. Thecomposition of claim 2, wherein said modified thermostable eubacterialDNA polymerase enzyme and said nucleic acid molecule and said targetnucleic acid are contained in a reaction mixture.
 4. The composition ofclaim 1, further comprising a buffer comprising magnesium.
 5. Thecomposition of claim 1, wherein said nucleic acid molecule comprises alabel.
 6. The composition of claim 5, wherein said label comprises afluorescent label.
 7. A composition comprising an isolated modifiedthermostable eubacterial DNA polymerase enzyme having 5′ nucleaseactivity but lacking polymerase activity, a first nucleic acid molecule,a second nucleic acid molecule, and a target nucleic acid, wherein saidtarget nucleic acid comprises a first portion downstream of a secondportion, wherein said first nucleic acid molecule comprises a regionthat is complementary to said second portion of said target nucleicacid, and wherein said second nucleic acid molecule comprises a regionthat is complementary said first portion of said target nucleic acid. 8.The composition of claim 7, wherein said first and said second portionsof said target nucleic acid are adjacent to one another.
 9. Thecomposition of claim 8, wherein said first nucleic acid moleculecomprises a label.
 10. The composition of claim 9, wherein said labelcomprises a fluorescent label.
 11. The composition of claim 1 or claim7, wherein said DNA polymerase is from a eubacterium of genus Thermus.12. The composition of claim 11, wherein said eubacterium is selectedfrom the group consisting of Thermus aquaticus, Thermus flavus, andThermus thermophilus.